Experimental Cell Research 249, 269–278 (1999)Article ID excr.1999.4442, available online at http://www.idealibrary.com on
Endogenous Interleukin 6 Conveys Resistance to cis-Diamminedichloroplatinum-Mediated Apoptosis
of the K562 Human Leukemic Cell Line1
George V. Z. Dedoussis,*,2 Athanasia Mouzaki,† Maria Theodoropoulou,† Panagiotis Menounos,*Marie-Christine Kyrtsonis,† Andreas Karameris,‡ and Alice Maniatis†
*Laboratory of Research, Nursing Military Academy, 16201 Vironas, Athens; ‡Department of Pathology, 401, Army General Hospital
of Athens, 11526 Athens; and †Laboratory of Haematology, University of Patras Medical School, Patras, Greece
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Cisplatin is an effective chemotherapeutic agenthat elicits its antineoplastic activity by binding toNA and disrupting template functions. IL-6 is a cyto-ine which has been shown to play a central role inost immunological defense mechanisms. Although562 leukemic cells have been shown to secrete IL-6,
ittle is known of whether there exists a correlationetween the expression of IL-6 and the resistance ofhese cells to anticancer chemotherapeutic agents. Toetermine the contribution of IL-6 to the regulation ofisplatin-induced apoptosis in K562 cells, we exam-ned whether treatment of K562 cells and cisplatin-esistant K562 subclones with anti-IL-6 mAb enhancesheir sensitivity to cisplatin. The results show thatis-diamminedichloroplatinum (CDDP) resistanceas overcome by treatment with nontoxic doses ofDDP in combination with anti-IL-6 mAb. When we
ested if the synergistic effect of anti-IL-6 and cisplatinould restore the ability of K562 mutant cells to un-ergo apoptosis, we found the typical DNA laddering
Cisplatin is an effective chemotherapeutic agent thatlicits its antineoplastic activity by binding to DNAnd disrupting template functions [1]. However, in
1 This work was supported by a Research Grant from the Depart-ent of Pathology, 401, Army General Hospital of Athens.2 To whom correspondence and reprint requests should be ad-
ressed at Xarokopio University, 70 El. Venizelou Str., 176 71 Kal-
ost patients who initially responded to cis-diam-inedichloroplatinum (CDDP), the tumor became un-
esponsive to the drug upon continuous treatment [2].IL-6, a cytokine with pleiotropic activities, has been
hown to play a central role in host immunologicalefense mechanisms [3]. Although the first detectedunction of IL-6 was the induction of B-cell differenti-tion and the production of immunoglobulins, IL-6 islso involved in other activating processes [4, 5]. Sev-ral studies have shown that IL-6 is a growth factor forarious tumors such as myeloma, AIDS Kaposi sarco-a-derived cells, and certain T- and B-cell lymphomas
6–8]. Apoptosis, a physiologically programmed event,s an active suicide process requiring energy-depen-ent participation of the dying cells [9]. Several anti-ancer chemotherapeutic agents such as CDDP inducepoptosis [10] and IL-6 suppresses the induction ofpoptosis by cytotoxic agents, transforming growth fac-or b1, and wild-type p53 [11, 12]. Although K562 andhronic myelogenous leukemia neoplastic cells haveeen shown to secrete IL-6 [13], little is known about aossible correlation between IL-6 expression and theesistance of these cells to anticancer chemotherapeu-ic agents. To determine the contribution of IL-6 to theegulation of drug-induced apoptosis in K562 cells, wenvestigated whether treatment of K562 cells andDDP-resistant K562 subclones, established in our
aboratory [14], with anti-IL-6 mAb enhances theirensitivity to anticancer chemotherapeutic agents.
MATERIALS AND METHODS
Reagents. CDDP was obtained from Bristol (Syracuse, NY). MTT,cridine orange (A-6014), and ethidium bromide (E-8751) were ob-ained from Sigma Chemical Company (St. Louis, MO). Rabbit anti-uman IL-6 antibodies (AHP254) were obtained from Serotec. The
sotype of the antibodies is IgG1. Anti-IL-6 mAb is capable of neu-ralizing IL-6 activity.
Cell lines. K562 erythroleukemic cells were cultured in RPMI640 medium supplemented with 10% heat-inactivated fetal calferum, penicillin (100 U/ml), streptomycin (100 mg/ml), L-glutamine
2 mM), and HEPES (10 mM) in a highly humidified atmosphere
ith 5% CO2 at 37°C. K562/B6 and K562/C9 are CDDP-resistant562 cells established in our laboratory by culturing K562 cells in
he presence of escalating doses of CDDP. Initial concentration ofDDP was 100 ng/ml and was increased to a maximum of 1 mg/ml.fter 3–4 weeks maintenance in medium containing CDDP, cellsere seeded into each well of a 96-well U-bottomed plate at a densityf 10 to 100 cells/well. When a well was observed to contain a largeolony, the colony was isolated and propagated into the resistantubline. Experiments with these sublines were performed 2–3 weeksfter removal of CDDP and maintenance in drug-free medium.Analysis of DNA fragmentation. For the analysis of the DNA
addering, characteristic of apoptotic cell death, DNA was isolatedrom 2 3 106 cells by the salting-out procedure previously described15]. Briefly, the cells were pelleted and digested overnight at 37°C inlysis buffer containing 10 mM Tris–HCl, pH 8.0, 2 mM EDTA, 400M NaCl, 0.6% (w/v) SDS, and 166 mg/ml proteinase K. After diges-
ion the salt concentration (NaCl) was raised to 1 M and the tubesere shaken vigorously. Samples were centrifuged. The superna-
ants were collected, and DNA was precipitated. The DNA wasesuspended in 25 ml of 10 mM Tris–HCl, 1 mM EDTA, pH 8.Nase-free RNase (1.5 units) was then added to the samples before
ncubation at 37°C for 2 h. After addition of loading buffer, theamples were subjected to electrophoresis on a 2% agarose gel in auffer containing 89 mM Tris–HCl, 89 mM boric acid, 2 mM EDTA,H 8, at 45 V for 16 h. A 123-bp DNA ladder was used as standard.NA was visualized by ethidium bromide staining (2 mg/ml, 1 h),estained overnight in redistilled water, and photographed underV illumination.Morphological assessment of apoptosis in target cells. At the end
f the incubation time with cisplatin, target cell pellets were col-ected on a glass slide, stained with 1 ml of a mixture of 100 mg/mlcridine orange and 100 mg/ml ethidium bromide in PBS–BSA 1%,nd immediately examined under a fluorescence microscope (Leitzaborlux K, Wetzlar, Germany) at a 490-nm excitation wavelength.Cytokine assay. The enzyme-linked immunosorbent assay
ELISA) kits were purchased from R&D Systems (Minneapolis, MN).he samples were prepared and used according to the recommenda-ions of the manufacturer. The detection limit of these assays is 4g/ml of each cytokine tested.Cytotoxicity assays. The cytotoxicity effect of cisplatin alone or in
ombination with rabbit anti-IL-6 antibody was assessed by trypanlue dye exclusion and by MTT. Briefly, the cells were resuspendedn complete medium at a concentration of 2 3 105 cells/ml. One
illiliter of cell suspension (K562; CDDP-resistant K562 subclones)as distributed into each well of 24-well culture plates (Costar), 1 mlf the appropriate dilutions of anti-IL-6 antibody was added to theells, and the cultures were incubated for 48 h. In all experiments,
wo different controls were used: cells without the addition of thenti-IL-6 antibody and cells with normal rabbit serum at a concen-ration comparable to the highest anti-IL-6 antibody concentrationsed. Cell viability was determined by the ability of cells to excluderypan blue. The MTT assay system used was essentially the same asreviously reported [16]. As a background value, a well containingnly RPMI–FCS plus MTT plus isopropanol was used. Each experi-ent was performed in triplicate. IC50 was defined as 50% reduction
n optical density in each test and fractional absorbance was calcu-ated using the formula [(mean absorbance in three test wells 2bsorbance in background well/(mean absorbance in three controlells 2 absorbance in background well)] 3 100.RNA extraction, first-strand cDNA synthesis, and PCR. Total
ellular RNA was isolated as described elsewhere [17]. The se-uences of the primers used are for IL-6, upstream 59-GCCAGAGCT-TGCAGATGAG, downstream 59-AGGAACTCCTTAAAGCTGCG,hich generate a 187-bp fragment, and for b2-microglobulin, up-
tream 59-TCACTCAATCCAAATGCGGC-39 which generates a
50-bp fragment [22]. For quantitative evaluation of the photo- a
raphs of the RT-PCR gels shown in Fig. 2, the bands were scannedsing a computing densitometer and the data analyzed using Image-uant V3.0 software, both from Molecular Dynamics (Sunnyvale,A).Flow cytometric analysis of P-glycoprotein expression. For label-
ng with the C219 anti-P-glycoprotein mAb, cells were fixed with 70%ethanol for 10 min at 220°C. Methanol-fixed cells were washedith PBS (4°C) and labeled with FITC-conjugated C219, a murine
IgG2a) monoclonal antibody specific for an intracellular epitope of-glycoprotein, an isotypic FITC-labeled control antibody, or unla-eled C219 antibody. P-glycoprotein expression was measured flowytometrically using a fluorescence-activated cell sorter analyzerBecton–Dickinson Immunocytometry Systems, Mountain View,A).GSH studies. After incubation for the appropriate time, the K562
ells and their CDDP-resistant subclones were centrifuged at 800gor 10 min. The supernatant was separated and the cells were re-onstituted with 0.5 ml of phosphate buffer (0.1 M PO4 containing.005 M EDTA, pH 7.4). After the samples were frozen and thawedhree times in a methanol/dry ice bath, they were centrifuged15,000g, 4°C) for 30 min. The supernatant was used to measureSH levels as previously described [23]. GSH levels were expresseds mg thiol g21 protein.Statistical analysis. Comparison between CDDP-resistant K562
ubclones and the cell line were determined by using the paired test. Differences between the means were considered statisticallyignificant at P , 0.05. Calculations of synergistic cytotoxicity wereetermined by using isobologram analysis as described by Beren-aum [33].
RESULTS
orphology and Growth Characteristics of K562 andCDDP-Resistant Subclones
The maternal K562 cells and the CDDP-resistant562 subclones have similar blastic morphology, asetermined by Wright stain, and demonstrate no ery-hroid differentiation as defined by negative benzidinetaining.
L-6 Secretion by K562 and Their CDDP-ResistantSubclones
We measured the IL-6 production by the K562 cells
FIG. 1. Time course of IL-6 secretion by K562 and their CDDP-esistant subclones K562/B6 and K562/C9 as measured by ELISA.06 cells were cultured in 24 wells for the indicated time points, theells were pelleted, and the supernatants were collected.
nd their CDDP-resistant subclones (K562/B6 and
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271IL-6 CONVEYS RESISTANCE TO CISPLATIN-MEDIATED APOPTOSIS
562/C9) into the culture medium, using a sensitiveLISA system. Both cell lines secrete IL-6 and the
evel of secretion was higher for the CDDP-resistantubclones than for the maternal cells. The time courseinetics of IL-6 accumulation into the medium re-ealed that IL-6 secretion increases continuously dur-ng the first 18 h for the maternal cells and 22 and 24 hor K562/B6 and K562/C9, respectively, after whichimes it reached a plateau (Fig. 1). At this time pointhe IL-6 concentration was 80 pg/ml for K562 cells and20 and 260 pg/ml for K562/B6 and K562/C9 cells,espectively. PCR analysis confirmed the above find-ngs by demonstrating IL-6 gene expression in the ma-ernal cells and the cisplatin-resistant cell lines (Fig.
FIG. 2. RT-PCR for the detection of IL-6 mRNA in the K562 cells.DNA samples correspond to 105 (A) and 103 (B) K562 cells per PCR.2 microglobulin RNA (C) was extracted from 103 cells (all lanes).W, molecular weight marker FX174 DNA/HinfI. Lanes 1 (both A
nd B), PCR negative control (no RNA in the reaction mix). Lanes 2both A and B), RNA extracted from 105 ex vivo peripheral bloodononuclear cells (negative control). Lanes 3 (both A and B), RNA
xtracted from 105 peripheral blood mononuclear cells cultured over-ight with 10 mg/ml PHA and 1 ng/ml PMA (positive control). Lanes, K562 cells. Lanes 5, K562 cells cultured with 0.1 mg/ml cisplatin.anes 6, K562 cells cultured with 1 mg/ml cisplatin. Lanes 7,562/B6 cisplatin-resistant subclone. Lanes 8, K562/C9 cisplatin-
esistant subclone. (D) Quantitative evaluation of the relative ex-ression levels of IL-6 mRNA measured by densitometry from B and
mxpressed as pixels.
). Reverse transcriptase PCR revealed the presence of187-bp fragment specific for IL-6 mRNA. In an at-
empt to elucidate any quantitative differences con-erning the levels of IL-6 mRNA among the cell lines,e extracted RNA from a reduced number of cells ande discovered that the amount of IL-6 mRNA from theaternal K562 cell line was less than that found in the
wo resistant cell lines (Fig. 2B, lanes 4, 7, and 8). Inddition we show that this important difference in theevel of IL-6 mRNA is due to the effect of cisplatin,ince the culture of K562 cells for 12 h with 0.1 and 1g/ml cisplatin significantly enhanced the mRNA levelf IL-6 in a dose-dependent manner (Fig. 2B, lanes 4, 5,nd 6). The effect of cisplatin in low doses to induceL-6 production is certified in Fig. 2D, in which byensitometry we show a significant increase of IL-6
FIG. 3. Time kinetics of combined cytotoxic activity of variousoncentrations of CDDP and anti-IL-6 mAb on K562, K562/B6, and562/C9 cells. Normal rabbit serum (}), anti-IL-6 mAb 1 mg/ml (E),DDP 10 mg/ml (h), CDDP 10 ng/ml (F), CDDP 10 ng/ml andnti-IL-6 mAb 1 mg/ml (X), CDDP 1 mg/ml and IL-6 1 mg/ml ({). The
of cytotoxicity was determined by the 4-h MTT assay. Points,eans of three different experiments performed in duplicate; bars,SD. *P , 0.05 compared with the CDDP 10 ng/ml alone.
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272 DEDOUSSIS ET AL.
isplatin and in both cisplatin-resistant K562 sub-lones.
ensitization of K562 Cells and Their CDDP-Resistant Subclones to CDDP by Anti-IL-6
In order to test if the elevated concentrations ofL-6 found in CDDP-resistant K562 subclones, com-ared to those of the maternal cells, correlated withhe diminished susceptibility of the mutant cells toisplatin cytotoxicity, we first examined the effect ofnti-IL-6 mAbs on the sensitivity of K562, K562B6,nd K562C9 to CDDP (Fig. 3). The results show thatDDP resistance was overcome by treatment withontoxic doses of CDDP (1 mg/ml) in combinationith anti-IL-6 mAb only after 4 days in culture.sing the same concentrations of CDDP and anti-
L-6 mAb, the maternal K562 cells demonstratedignificant differences (compared to the addition ofDDP alone) in the percentage of survival even afterdays in culture. In contrast, there was no signifi-
ant change in the percentage of cell viability whene added to the cultures anti-IL-6 alone, suggesting
hat anti-IL-6 had a predominant cytostatic effect onhese cell lines. To obtain similar results with 100-old less concentration of cisplatin 10 ng/ml and the
FIG. 4. Synergistic interaction of anti-IL-6 antibody and CDDP.n the presence of the different combinations of CDDP with the anti-f three different experiments. *P , 0.05 versus CDDP alone. CDDnti-IL-6 mAb at 1 mg/ml ({), CDDP with anti-IL-6 mAb at 10 mg/m
ame concentration of anti-IL-6 mAb, a longer period l
f culture (4 days) was necessary for the K562 cells,hereas the same combination of agents did notrovoke significant cytotoxicity in the CDDP-esistant subclones. Anti-IL-6 mAbs by themselvesid not inhibit cell proliferation in a 4-day MTTssay (data not shown). In order to find the loweroncentration of CDDP which, in combination withhe appropriate concentration of anti-IL-6 mAb,ould result in the same level of cytotoxicity inDDP-resistant K562 cell lines as was observed with
he toxic doses of CDDP alone, we measured theytotoxicity at different combinations of the twogents after 4 days in culture. Clearly the combina-ion giving levels of cytotoxicity similar to those of 10g/ml CDDP alone was that of 100 ng/ml cisplatinnd 10 mg/ml anti-IL-6 mAb (Fig. 4).
nduction of Apoptosis
Cross-resistance to unrelated drugs is an expectedroperty of mutant cells altered in the process of apo-tosis. The ability of K562 and K562/C9 to undergopoptosis was initially examined by analysis of DNAragmentation in cells exposed to equitoxic levels ofisplatin (Fig. 5). K562 cells were exposed for 72 h toither 1 mM (Fig. 5, top, lane d) or 10 mM (Fig. 5, top,
percentage of cytotoxicity was measured after 4 days of cell culture6 mAb in a 4-h MTT assay. The results are expressed as the meanslone (h), CDDP with anti-IL-6 mAb at 0.1 mg/ml (E), CDDP with).
TheIL-P a
ane c) cisplatin, which corresponded to the ID50 and
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273IL-6 CONVEYS RESISTANCE TO CISPLATIN-MEDIATED APOPTOSIS
D90, respectively. As shown in Fig. 5, DNA cleavageith a characteristic pattern of internucleosomal lad-
FIG. 5. Analysis of drug-induced internucleosomal DNA cleav-ge in K562 and in K562/C9 cells. (Top) K562 cells were exposed for2 h to either 1 (lane d) or 10 mM (lane c) cisplatin, which corre-ponds to the ID50 and ID90, respectively. DNA cleavage with aharacteristic pattern of internucleosomal ladder (multiples of ap-roximately 180-bp fragments) was observed in these cells exposedo the lower or the higher cisplatin concentrations. In contrast in562/C9 cells exposed to 3.3 (lane a) or 33 mM (lane b), corresponding
o ID40 or ID90, respectively, no apoptotic pattern was observed. Lane, 123-bp ladder marker. (Bottom) Induction of DNA oligonucleoso-al pattern by anti-IL-6 mAb in combination with cisplatin on K562
nd their CDDP-resistant K562 subclone. Lane 1, K562 cells afterynergistic interaction of anti-IL-6 10 mg/ml and CDDP 100 ng/ml;ane 2, K562 cells cultured with CDDP 100 ng/ml; lane 3, K562/C9ells after synergistic interaction of anti-IL-6 10 mg/ml and CDDP 1g/ml; lane 4, K562/C9 cells cultured with 1 mg/ml cisplatin.
er (multiples of approximately 180-bp fragments) was g
bserved in these cells exposed to the lower or theigher cisplatin concentrations. At the higher concen-ration a small smear appears after the laddering,hich could be explained by a low percentage of cellsndergoing necrosis. In contrast in K562/C9 cells ex-osed to 3.3 mM (Fig. 5, top, lane a) or 33 mM (Fig. 5,op, lane b), corresponding to ID40 or ID90, respectively,o apoptotic pattern was observed. Moreover, somemearing appeared on the gel at the high doses, indi-ating random continuous degradation of cellularNA, a process which characterizes necrosis [19].Previous reports have implicated IL-6 as a resis-
ance-promoting factor against CDDP for various cellines [23, 24]. We thus examined the effect of IL-6eutralization on K562 and K562/C9 cell sensitivity tondergoing apoptosis after treatment with cisplatinnd anti-IL-6 polyclonal antibodies. As we can see inig. 5, bottom, when the K562 cells and their CDDP-esistant cell line were incubated for 4 days continu-usly with 100 ng/ml and 1 mg/ml cisplatin, respec-ively (lanes 2 and 4), no apoptotic pattern wasbserved. When cisplatin was used in combinationith 10 mg/ml anti-IL-6 antibody, a typical apoptoticattern was observed on both cell lines (lanes 1 and 3).t is noteworthy that DNA laddering was achieved atoncentrations of cisplatin 100-fold less than those re-uired to achieve similar cytotoxicity by the drug usedlone. Thus the synergistic effect of anti-IL-6 and cis-latin restored the ability of K562 mutant cells tondergo apoptosis even in the presence of a nontoxicose of the drug.DNA fragmentation is not the sole criterion used to
efine apoptosis. Originally this process was identi-ed using morphological criteria [20]. One of thearliest morphological events in apoptotic cells ishromatin condensation to the nuclear membrane.luorescence microscopy was used to morphologi-ally characterize cells exposed to cisplatin and sub-equently stained with acridine orange (AO) andthidium bromide (EtBr). Both AO and EtBr inter-alate DNA and stain nuclei green and orange, re-pectively. AO stains both viable and nonviable cellsreen, whereas EtBr stains the nonviable cells or-nge. Figure 6 shows that the DNA degradation,bserved in K562 and K562/C9 cells exposed for 72 ho cisplatin or cisplatin and anti-IL-6 antibody, wasssociated with modifications in the chromatin dis-ribution. Nuclei of control cells were rounded andontained homogeneously dense stained chromatinFigs. 6A and 6B). Cisplatin-treated K562 cells con-ained clumps of fragmented chromatin and a num-er of cells appeared dead (i.e., stained orange) (Fig.C). CDDP-resistant cells exposed to cisplatin aloneetained a well-defined plasma membrane in agree-ent with EtBr exclusion and their nuclei, in their
reat majority, had a normal morphology (Fig. 6D).
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274 DEDOUSSIS ET AL.
n the other hand when both cell lines were culturedn presence of nontoxic doses of CDDP and anti-IL-6ntibody (10 mg/ml) apoptotic bodies and cell shrink-ge appeared (Figs. 6E and 6F). The DNA fragmen-ation observed in treated cells clearly preceded theoss of plasma membrane integrity. This prolonged
aintenance of membrane integrity has been re-orted to be a convenient criterion for distinguishing
FIG. 6. Fluorescent microscopy of K562 and K562/C9 cells. Cellsncubated with cisplatin (1 mM for K562 and 3.5 mM for K562/C9; C) and then stained with AO and EtBr (72 h). The arrows point to
dead).
poptotic cell death from necrosis [21]. i
echanism of Sensitization to CDDP byAnti-IL-6 mAb
We then asked whether anti-IL-6 mAbs affect theultiple drug-resistant (MDR) phenotype on both562 cells and their CDDP-resistant counterparts. Thexpression of P-glycoprotein by flow cytometry wasnhanced on the two cisplatin-resistant K562 cell lines,
re either stained immediately with AO and EtBr (A and B) or wereD, respectively) or cisplatin and anti-IL-6 10 mg/ml for 72 h (E and
s with apoptotic morphology, stained either green (alive) or orange
weandcell
ndicating that the MDR phenotype could be partially
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275IL-6 CONVEYS RESISTANCE TO CISPLATIN-MEDIATED APOPTOSIS
esponsible for the diminished susceptibility of theseells to cisplatin-induced cytotoxicity. On the otherand when the same cell lines were incubated withnti-IL-6 antibody it was revealed that the P-lycoprotein expression was not modified by the anti-ody treatment, suggesting that the MDR phenotype isot directly involved in sensitization (Table 1). We thennalyzed the GSH levels in the maternal cells and theirDDP-resistant subclones with or without the additionf cisplatin or anti-IL-6 mAbs. The cells were starvedor 2 days before the experiment in order to achieveynchronization. As we can see in Fig. 7 the K562/C9ells produced more GSH than the maternal cell line.hen both cell lines were cultured in the presence of
isplatin the thiol increased especially for the K562ells. In contrast treatment with anti-IL-6 mAb down-
FIG. 6—
TABLE 1
Effect of Anti-IL-6 mAb on the Expression ofP-Glycoprotein on K562, K562/B6, and K562/C9 Cells
Note. K562, K562/B6, and K562/C9 cells were preincubated withedium, control antibody (10 mg/ml), or anti-IL-6 mAb (10 mg/ml) for
2 h and then washed and tested for the expression of P-glycoprotein.
ta Means 6 SD of three different experiments.
egulated the GSH levels. These results suggest thathe reduction of GSH levels may be partially responsi-le for the sensitization of cisplatin-resistant K562ells by anti-IL-6 mAb.
DISCUSSION
The present study demonstrates that antibody-me-iated neutralization of IL-6 secretion by CDDP-resis-ant K562 subclones restores their diminished suscep-ibility to programmed cell death by CDDP. Thesetudies demonstrate that neutralization of protectiveactors produced by the tumor cells can reverse drugesistance of tumor cells. The enhanced sensitivity wasbtained in the parental K562 cells and the CDDP-esistant K562/B6 and K562/C9 subclones. These cis-latin-resistant cell lines were found to be less suscep-ible to NK cells isolated from healthy donors or fromatients with breast cancer [14]. By using a humanrythroleukemic cell line it was previously suggestedhat an important element in MDR was reduced sus-eptibility to lysis by natural killer cells [26]. Thisssociation between the development of multidrug re-istance and natural killer resistance supports clinicalbservations of rapid tumor progression in patientsho have failed initial chemotherapy. Recently, it haseen shown that diverse anticancer drugs (includingisplatin) may exert their toxicity through the induc-ion of apoptotic cell death [10]. Apoptosis is also foundn target cells attacked by cytotoxic T lymphocytes andatural killer cells [27]. Since apoptosis contributes to
ntinued
he toxicity of anticancer drugs, an attractive hypoth-
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276 DEDOUSSIS ET AL.
sis is that some cell lines acquire drug resistance byltering this process [28].The data obtained from our cell culture system are in
ccordance with published data claiming that a func-ional pathway for apoptosis contributes to the drugusceptibility of wild-type cells and that this process isltered in the mutant cells; the selection for cisplatinesistance resulted in the selection of a defective apop-otic process. This strongly suggests that alterations ofhis process could be a mechanism of drug resistance.t has been demonstrated that IL-6 suppressed thenduction of apoptosis induced by anticancer chemo-herapeutic agents [12]. It has been also reported thatL-6 is a viability factor for both normal and leukemicells [34]. K562 cells and their cisplatin-resistant sub-lones were found both to express IL-6 mRNA and toecrete active IL-6 protein. The higher levels of IL-6RNA detected in the two K562 cisplatin-resistant
ubclones could explain the elevated concentrations ofL-6 protein. This induction of IL-6 gene transcriptionnd transduction could be attributed to the effect ofDDP, since we demonstrated that after a short periodf culture of K562 cells with different doses of cisplatin,clear enhancement of IL-6 mRNA was noticed. In
ddition to cisplatin, other antineoplastic agents likeiazofurin [13] stimulate K562 cells to increase IL-6roduction.Our findings suggest that treatment with anti-IL-6Ab enhanced the sensitivity of K562 cells to CDDP by
locking IL-6-mediated inhibition of the induction ofpoptosis. Synergy was achieved at suboptimal concen-rations of each agent, much lower than those requiredo obtain similar cytotoxicity by either drug used alone.
The interest in apoptosis in oncology stems from thebservation that at least in some tumors, malignancy
FIG. 7. GSH content of K562 and K562/C9 cells without drug exp562/C9) or cisplatin and anti-IL-6 10 mg/ml for 72 h. Both cell line
ay be associated with the loss of the ability of the cell l
o undergo spontaneous apoptosis rather than withncreasing cell proliferation rates [29].
One of the mechanisms associated with cisplatin re-istance is the expression of the mdr-1 gene product,he P-glycoprotein. Several reports [30, 31] describe anverexpression of the P-170 in cell lines of differentrigin after treatment with cisplatin. In accordanceith these data we found that compared to the mater-al cells, a greater number of CDDP-resistant cellsxpressed the P-glycoprotein antigen. CDDP may se-ect the P-glycoprotein subpopulation and may alsonduce CDDP resistance. The P-glycoprotein-mediatedhenotype, therefore, can coexist in resistant tumorells having primary resistance to CDDP. It has beenhown [32] that a majority of P-glycoprotein-positiveDR human leukemia subclones displayed slight but
tatistically significant resistance to endogenousnd/or interferon-activated NK-cell-mediated lysis,ompared to the drug-sensitive maternal type; there-ore we could also ascribe the enhanced resistance toK-mediated cell death (data not shown) to the MDRhenotype. Previous results have shown that the classi GST is the dominant isoenzyme for cytosolic gluta-
hione transferases in K562 cells [35]. One of the func-ions of GST-p is to conjugate detoxification alkylatinggents to glutathione and as has been demonstrated,ST-p is down-regulated in renal carcinoma cells fol-
owing treatment of the tumor cells with anti-IL-6 mAb25]. Glutathione, a tripeptide thiol, rises in some cis-latin-resistant cancer cells and plays a primary role inellular detoxification of electrophilic compounds [36].t is the most abundant nonprotein sulfydrylic com-ound in mamalian cells, including K562 cells [37]. Thessociation of IL-6 with GSH levels and with protectiongainst alkylating agents has been previously pub-
ure or after treatment with cisplatin (1 mM for K562 and 3.5 mM forere starved for 2 days before each experiment.
os
ished [38]. We found elevated the levels of GSH in the
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277IL-6 CONVEYS RESISTANCE TO CISPLATIN-MEDIATED APOPTOSIS
isplatin-resistant K562 cells, and when we treatedhese cells with anti-IL-6 mAb, there was a significantecrease of thiol content. Therefore we could partiallyttribute the sensitization to cisplatin of our K562isplatin-resistant cells after the combined action withnti-IL-6 mAbs to the reduction of GSH levels.The current studies showed that anti-IL-6 mAb sen-
itized CDDP-resistant K562 cells to CDDP by induc-ion of apoptotic death. The synergy obtained with lowoncentrations of each drug is of clinical relevance,ince high concentrations of the drugs used alone areoxic in vivo. Consequently, the therapeutic use ofDDP in combination with anti-IL-6 mAb may proveseful in the treatment of patients with CDDP-resis-ant cancer cells.
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eceived September 7, 1998evised version received February 15, 1999
