Growth Inhibition, Cytokinesis Failure and Apoptosis of Multidrug-Resistant

Home , Cytokinesis

Leukemia (1999) 13, 768–778  1999 Stockton Press All rights reserved 0887-6924/99 $12.00 http://www.stockton-press.co.uk/leu Growth inhibition, cytokinesis failure and apoptosis of multidrug-resistant leukemia cells after treatment with P-glycoprotein inhibitory agents G Lehne1,2, P De Angelis3, M den Boer4 and HE Rugstad1

1Department of Clinical Pharmacology, 2Institute for Surgical Research, and 3Institute for Pathology, The National Hospital, Rikshospitalet, University of Oslo, Norway; and 4Department of Pediatrics, Free University Hospital, Amsterdam, The Netherlands

The multidrug transporter P-glycoprotein (Pgp), which is fre- inhibitors with less toxic potential have been developed quently overexpressed in multidrug resistant leukemia, has including the non-immunosuppressive cyclosporin SDZ PSC many proposed physiological functions including involvement 833,12 the cyclopeptolide SDZ 280–446,13 and the cycloprop- in transmembraneous transport of certain growth-regulating 14 cytokines. Therefore, we studied cell growth of three pairs of yldibenzosuberane LY 335979. These agents exercise high drug resistant and sensitive leukemia cell lines (KG1a, K562 afﬁnity for Pgp and interfere with binding of known substrates and HL60) exposed to three different inhibitors of Pgp. The such as azidopine and calcein.15–17 SDZ PSC 833 which is resistant KG1a and K562 sublines, which expressed high levels the most extensively studied of these, has proven to be well of Pgp, responded to low doses of the cyclosporin SDZ PSC tolerated in phase I trials,18–20 and is currently being investi- 833, the cyclopeptolide SDZ 280–446, and the cyclopropyldibenzosuberane LY335979 with a dose-dependent growth inhi- gated as an adjunct to cancer chemotherapy in phase II and bition. In the resistant variants of KG1a and K562 cells the III trials.

mean half-maximal growth inhibitory doses (GI50) of SDZ PSC Although Pgp contributes to renal and hepatic elimination 833 were 312 (SE 41) and 414 (SE 50) nM, those of SDZ 280– of toxic compounds,21 adrenal secretion of steroid hormones22 446 were 685 (SE 51) and 578 (SE 54) nM, and those of LY335979 and tissue protection at the blood–brain barrier,23 the physio- ␮ were 66 (SE 1) and 48 (SE 8) nM, respectively. Exposure to 1 M logical role of Pgp is not fully understood. Two recent reports SDZ PSC 833 resulted in tetraploidization, cytokinesis failure have shown that Pgp may also take part in the transport of and apoptosis of the KG1a and K562 MDR variants. Conversely, 24,25 parental cells with no or low levels of Pgp and the non-Pgp growth stimulating cytokines IL-2 and IL-4. Thus, the par- resistant variant of HL60 cells were not receptive to these cyto- ticipation of Pgp in growth regulation could represent a new toxic effects. We conclude that inhibition of Pgp may exercise target for growth control in neoplastic cells expressing Pgp. selective cytotoxicity in Pgp-rich leukemia cells indicating a Recently, we discovered a selective cytotoxic effect of SDZ possible therapeutic target in multiresistant leukemia. PSC 833 which engaged apoptosis (programmed cell death) Keywords: P-glycoprotein; apoptosis; leukemia; resistance modu- 26 lator in multidrug resistant leukemia cells. As this effect appeared to involve Pgp, the present paper addresses the effect of other modulators on both Pgp- and non-Pgp-expressing leukemia cells, and further focuses on the induction of apoptosis in Introduction relation to cell cycle progression. The cells were characterized with respect to MDR phenotype and expression of proteins Failure to achieve complete and durable responses from can- involved in the promotion (p53, Bax, Apol/Fas) and sup- cer chemotherapy is a common clinical problem that limits pression (Bcl-2, Bcl-xL) of apoptosis. Pgp overexpression was the curative potential of anti-cancer drugs in clinical the only common denominator which was exclusively asso- oncology. Multidrug resistance (MDR) is believed to be a ciated with a dose-dependent growth inhibitory response to major cause for treatment failure, and is frequently associated the modulators SDZ PSC 833, SDZ 280–446, and LY335979. with overexpression of the multidrug transporter, P-glyco- Furthermore, the KG1a and K562 MDR variants, which shared protein (Pgp), which is an integral plasma membrane protein these characteristics, also demonstrated attributes of cytokin- capable of drug expulsion and maintenance of tolerable intra- esis failure and apoptosis after exposure to the modulators. cellular levels of certain cytotoxic drugs.1 Several clinically important anti-cancer drugs may be removed from neoplastic cells by Pgp-mediated transport, despite the diversity in Materials and methods chemical structures and mechanisms of action.2 This ability is apparently reflected in a negative correlation between Pgp Chemicals expression and chemosensitivity or survival in leukemias,3,4 lymphomas5 and some solid tumors.6–8 The human leukemia cell lines were propagated in RPMI 1640 Pgp has a broad specificity for substrates, and several non- medium with l-glutamine (BioWhittaker, Walkersville, MA, cytotoxic drugs may competitively inhibit efflux of cytotoxic USA) supplemented with 10% fetal calf serum, streptomycin drugs by Pgp and thereby downmodulate MDR.9 The calcium (100 ␮g/ml), penicillin (100 U/ml), nystatin (40 U/ml) and channel blocker verapamil was the first agent that was shown HEPES (only K562 cell lines). MRK16 was kindly provided by to modify MDR in vivo and in vitro,10 but unfortunately the Professor T Tsuruo (Institute of Molecular and Cellular Biosci- MDR modulating activity required concentrations that are ences, The University of Tokyo, Japan), and UIC2 was pro- associated with severe cardiac toxicity in patients.11 There- vided by Immunotech (Marseille, France). MRPr1 was a gift fore, a new generation of highly specific and potent Pgp from Dr MJ Flens and Professor RJ Scheper (Department of Pathology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands). The IgG2a isotypic control antibody Correspondence: G Lehne, Department of Clinical Pharmacology, Rikshospitalet, The National Hospital of Norway, N-0027 Oslo; Fax: MOUSE IgG2a was purchased from Monosan (Uden, The 47 22 11 19 87 Netherlands). SDZ PSC 833 and SDZ 280–446 were supplied Received 28 September 1998; accepted 28 December 1998 by Novartis (Basel, Switzerland). LY335979 and vincristine Selective cytotoxicity of P-glycoprotein inhibitory agents G Lehne et al 769 were supplied by Eli Lilly & Co. (Indianapolis, IN, USA). Dau- described.31 Determination of MRP required fixation in 0.74% norubicin was provided by Rhoˆne Poulenc Rorer (Vitry, v/v formaldehyde in acetone for 10 s at room temperature to France). allow staining of a cytoplasmic epitope with MRPr1 (1.7 ␮g/ml) antibody as previously described.32 Immunofluo- rescence distributions were generated using a FACScan flow cytometer (Becton Dickinson, San Jose´, CA, USA) tuned to Cells and culture conditions 488 nm laser excitation wavelength. FITC fluorescence of gated populations was collected through a bandpass filter Multidrug-resistant human acute myelogenous leukemia cell (FL1, bandwidth 515–545 nm). Calculations of logarithmically lines were obtained by subculturing American Type Culture 27 28 amplified fluorescence values were performed in arithmetic Collection (ATCC) lines KG1a and HL60 in stepwise mode using the CellQuest computer program (Becton increased concentrations of daunorubicin and vincristine from Dickinson). Reproducibility was ensured by at least two repli- 10 to 100 ng/ml (KG1a/200) and from 2 to 65 ng/ml cate experiments. (HL60/130). The parental cells were designated KG1 a/0 and HL60/0, respectively. The human chronic myelogenous K562 (ATCC) cell line29 designated K562/0, and a multistep selected MDR subline (K562/150) adapted to 150 nm vincristine30 were Intracellular accumulation of daunorubicin: The cells provided by Dr Astrid Gruber (Karolinska Hospital, Stock- were grown in drug-free medium for 24 h prior to flow cyto- holm, Sweden). The cells were maintained and propagated metric analysis. A FACScan flow cytometer was used to gener- as previously described.31 The number of doublings (n) were ate daunorubicin fluorescence which provides excitation calculated according to the equation: maximum at 490 nm and emission maximum at 592 nm.33 The fluorescence was transmitted through a bandpass filter of n = 3.32 · log (X/X ) 0 564–606 nm (FL2) and logarithmically amplified. Correlated

where X0 is the cell count at seeding time point and X is the forward angle (a relative measure of cell size) and right angle cell count on day x of the culture. (a measure of cell granularity) light scatter measurements were generated to exclude dead cells and debris from analyses. Acquired data from 10000 events were analyzed using Cell Quest. By virtue of daunorubicin fluorescence intracellular Cell growth inhibition assay accumulation of the drug was measured as fluorescence distributions of cell samples after 1 h incubation at 37°C with × 4 Approximately 5 10 cells were plated in 16 mm-diameter 4.4 ␮m daunorubicin alone or in the presence of SDZ PSC wells (Costar Corporation, Cambridge, MA, USA) and grown 833, SDZ 280–446, or LY335979 at 1 ␮m concentrations. in 1 ml of drug-free medium for the first 24 h. The wells were Each experiment was performed three times. then supplemented with the drug or combination of drugs in a certain dose range. At least three replicate cultures were made for each of the dose levels and for untreated controls. The KG1a cells were cultured for 96 h, and the K562 and TUNEL assay for detection of apoptotic cells: Terminal HL60 cells were cultured for 72 h ensuing two to three doub- deoxynucleotidyl transferase(TdT)-mediated d-uridine-5Ј- lings. The actual mean numbers of doublings (standard error triphosphate(dUTP)-biotin nick end-labeling (TUNEL) utilizes of the mean in parenthesis) for KG1a/0 were 3.1 (SE 0.2), that the ability of the enzyme TdT to catalyze binding of deoxynu- for KG1a/200 were 2.7 (SE 0.2), that for K562/0 were 3.3 (SE cleotides to 3Ј-OH ends of DNA strand breaks. The method 0.2), that for K562/150 were 3.1 (SE 0.4), that for HL60/0 were used in this study is based on the method of Gorczyca et al34 3.3 (SE 0.1), and that for HL60/130 were 2.0 (SE 0.04). Har- and modified as described.26 Briefly, the cells were cultured vested cells were counted in a Coulter Counter ZM (Coulter for 48 h in the presence of 1 ␮m SDZ PSC 833, SDZ 280–446 Electronics, Luton, UK). Cell numbers were assessed elec- or LY335979, and then 106 cells were fixed in 1% ice-cold tronically with a lower threshold set at 7.5 ␮m particle diam- paraformaldehyde and subsequently in methanol at −20°C eter to discard cell debris and shrunken (apoptotic) cells. The before 30 min incubation at 37°C in a TdT solution

dose level required for 50% inhibition of cell growth (GI50) (Boehringer Mannheim, Mannheim, Germany) which was calculated from linear plots of dose vs cell number. The included biotin-labeled dUTP. Subsequently, the cells were resistance factor (RF) was defined as the ratio between the incubated on ice with streptavidin-fluorescein isothiocyanate ␮ GI50 values obtained in the resistant and sensitive cells, (FITC) for 30 min, and finally counterstained with 5 g/ml pro- respectively. By co-incubation with either SDZ PSC 833, SDZ pidium iodide (PI) to measure DNA content. Analyses were 280–446 or LY335979 modulation of growth inhibition was performed using FACScan and FACSVantage (Becton assessed. The modulating factor (MF) was defined as the ratio Dickinson) flow cytometers. FITC green fluorescence rep-

between the GI50 values of multidrug resistant cells with and resented DNA strand breaks as in apoptosis, and PI red ﬂuor- without the modulating agent, respectively. Each experiment escence represented DNA content. Doublet discrimination was performed at least three times. and exclusion of debris and clumps were achieved using red ﬂuorescence pulse-width analyses. Each experiment was performed three times. Flow cytometry

Determination of MDR phenotype: Pgp immunofluoresc- APO-1/Fas expression: The IgG2a antibody BMS138 and ence was obtained in viable cells by a three-layer staining the IgG2b antibody BMS140 (both Bender MedSystems, technique using MRK16 (20 ␮g/ml) and UIC2 (20 ␮g/ml) anti- Vienna, Austria) were selected as primary APO-1/Fas antibodies to reveal surface epitopes of Pgp as previously bodies. Cell suspensions were washed with PBS/BSA and Selective cytotoxicity of P-glycoprotein inhibitory agents G Lehne et al 770 incubated on ice for 60 min with BMS138 (10 ␮g/ml), Results BMS140 (10 ␮g/ml), MOUSE IgG2a (25 ␮g/ml), or MOUSE ␮ IgG2b (25 g/ml) in PBS/BSA. The second and third layer MDR phenotype staining protocols were carried out as described for Pgp detection and followed by flow cytometry as described in ‘Determi- nation of MDR phenotype’. Reproducibility was ensured by The MDR phenotypes were determined by flow cytometric two replicate experiments. immunofluorescence detection using anti-Pgp (MRK16, UIC2) and anti-MRP (MRPr1) antibodies. The degree of protein expression was determined by the expression factor (EF), which was defined as the ratio between mean fluorescence Cytospin preparations values for relevant (numerator) and irrelevant (denominator) antibody. To distinguish between wild-type and MDR phenotype a discrimination factor (DF) was defined as the ratio Cytospin preparations were made by centrifugation of aliquots between the EF values for resistant (numerator) and parental of approximately 105 cells in 300 ␮l RPMI and 300 ␮l fixative (denominator) cells. Using MRK16 the KG1a/200 and (95% methanol and 5% carbowax 1540) at 1000 r.p.m. for K562/150 cells were highly positive for Pgp with EF values of 5 min using Shandon Cytospin 2 (Shandon Scientific, 65.5 and 71.9, respectively. The parental KG1a/0 cells were Cheshire, UK). Automated Papanicolaou staining was per- slightly Pgp-positive (EF = 8.8) and the parental K562/0 cells formed in a Jung Autostainer XL (Leica, Germany) as were completely negative (EF = 0.7). Both the HL60/0 and previously described.26 HL60/130 cells expressed negligible levels of Pgp (EF = 2.9 and 1.6, respectively). In comparison with parental cells the resistant KG1a and K562 variants overexpressed Pgp multifold (DF = 7 and 103, respectively), whereas Pgp expression was Immunoblotting not increased in the resistant HL60 variant (DF = 0.6). These results were reproduced using UIC2 which resulted in corresponding DF values (5, 69 and 0.5, respectively). Over- Cell lines which were to be analyzed for expression of several expression of MRP occurred only in the resistant HL60 subline proteins involved in the regulation of apoptosis were prepared (DF = 5) using MRPr1. Only trace amounts of MRP were for immunoblotting as follows: 2–3 × 106 cells from each cell detected in the other leukemia cell lines of this study. line, washed once in PBS, were boiled for 5 min in sodium dodecyl sulphate (SDS) sample buffer containing 0.1 mm phe- nylmethylsulfonylfluoride (PMSF). These were then cooled on − ° ice and frozen at 20 C until use. The proteins and Kaleido- Pgp function scope molecular weight standards (BioRad, Hercules, CA, USA) were first separated by electrophoresis in 8.0% or 12.0% 39 SDS-polyacrylamide gels.35 Immunoblotting was done as Cytotoxic daunorubicin is a known substrate of both Pgp 40 described previously36 using the following antibodies: anti- and MRP. Therefore, we assessed Pgp or MRP function in Bax P-19 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) terms of cellular accumulation of daunorubicin using flow at final concentration 2 ␮g/ml, anti-Bcl-xL S-18 (Santa Cruz cytometry and fluorescence detection in cells that were incu- ° ␮m Biotechnology) at 2 ␮g/ml, anti-Bcl-2 clone 124 (Dako, Oslo, bated for 60 min at 37 C with 4.4 of the drug. Accumu- Norway) at 5 ␮g/ml, anti-p53 Pab 1801 (Calbiochem, Cam- lation of daunorubicin was restricted in all the resistant sub- р bridge, MA, USA) at 2.5 ␮g/ml and anti-p-83 34C1 (kindly lines compared to the corresponding wild types (P 0.02). By provided by Dr T Stokke, The Norwegian Radium Hospital, combining data from triplicate experiments with concurrent ␮m Oslo, Norway) at 1:30 dilution of stock. The amount of p83 exposure to 1 of SDZ PSC 833, SDZ 280–446 or was determined on the same blots as a control for gel loading LY335979, none of which were fluorescent, Pgp function and Ͻ and cell concentration as described previously.36 An amplified modulator activity were demonstrated (P 0.0001). The com- biotin-streptavidin alkaline phosphatase staining procedure bined fluorescence values (mean channel fluorescence) of (BioRad) was used to detect primary antibodies. Reproduci- KG1a/200 cells increased from 10.5 (SE 0.03) to 38.5 (SE 1.1), bility was ensured by two replicate experiments. that of KG562/150 cells from 18.2 (SE 0.5) to 37.7 (SE 0.4), and that of parental KG1a/0 cells from 17.4 (SE 0.9) to 27.9 (SE 0.7). The minor modulator activity in the KG1a wild type was due to a low constitutive Pgp expression as previously Statistics demonstrated. SDZ PSC 833 and SDZ 280–446 contributed equally to enhance the accumulation of daunorubicin in MRP expressing Comparisons of mean values were performed by two-tailed HL60/130 cells as treatment with these modulators increased unpaired t tests using Prism software (GraphPad, San Diego, the combined fluorescence values from 38.8 (SE 1.9) to 64.1 CA, USA). P values Ͻ0.05 were regarded as statistically sig- (SE 3.5) (P = 0.002). Conversely, LY 335979 did not contribute nificant. Summary statistics were reported as means provided to any change in daunorubicin content of cells with no or with standard errors of the mean (SE) or 95% confidence inter- very low levels of Pgp as demonstrated by stable fluorescence vals (CI). Histogram comparisons were performed by Kolmo- values of 33.9 (SE 0.8) and 34.4 (SE 1.1) in K562/0 cells (NS), gorov–Smirnov statistics37 to generate D values ranging of 85.4 (SE 5.2) and 79.4 (SE 6.8) in HL60/0 cells (NS), and between 0 and 1.0 according to the difference between two of 38.8 (SE 1.9) and 32.9 (SE 3.4) in HL60/130 cells (NS). distributions (higher values indicate greater difference). D These findings indicate LY335979 exclusively blocks the func- values у0.15 were regarded as significant differences tion of Pgp, whereas SDZ PSC 833 and SDZ 280–446 block between two histograms.38 the function of both Pgp and MRP. Selective cytotoxicity of P-glycoprotein inhibitory agents G Lehne et al 771 Cytotoxic effect of daunorubicin observed following treatment with SDZ 280–446 and LY335979 as well (Figure 4), and by using these agents the A measure of cytotoxicity was provided by determination of G2/M populations increased to 65% and 60%, respectively. the drug dose that reduced the cell numbers in continuously These populations were shown to consist of true tetraploid growing cultures by 50% (GI50). There was a 3-, 13- and 17- cells and not doublets or clumps, by the use of pulse-pro- fold relative resistance to daunorubicin in the resistant sub- cessed red fluorescence signals, which allowed for doublet lines of KG1a, K562 and HL60 cells, respectively. Low-dose discrimination. Correspondingly, microscopic images demon- treatment with SDZ PSC 833 (120 and 210 nm), SDZ 280–446 strated several binucleated cells as well as nuclear fragmen- (420 and 340 nm), or LY 335979 (40 nm) sensitized the MDR tation (Figure 5). The wild-type KG1a cells were not receptive variants KG1a/200 and K562/150 6.5-fold (SE 0.3) and 3.6- to these changes in cell cycle distribution. fold (SE 0.5), respectively, to the cytotoxic effect of daunorub- The cell cycle distribution of the resistant K562 cells icin. This substantial circumvention of MDR was in agreement underwent similar changes as the resistant KG1 a cells after with the highly significant increment of daunorubicin exposure to the three modulators. In particular, the changes accumulation in these cells following combined treatment induced by LY335979 were pronounced with reduction of the with anthracycline and modulator. In the MRP positive G1 phase population from 49% to 4%, reduction of the S HL60/130 cells SDZ PSC 833 and SDZ 280–446 reduced the phase population from 19% to 6%, increment of the G2/M mean GI50 value for untreated cells from 445 (SE 19) nm to phase population from 26% to 42% and emergence of large 220 (SE 41) nm, but the effect was obtained within a higher populations with subdiploid (23%) and polyploid (25%) cells. dose range (412–1235 nm). Apparently, LY335979 (1 ␮m) did The cell cycle distribution of the wild-type K562 cells not exercise any effect on the resistance of the HL60/130. remained unchanged after equivalent treatment.

Cytotoxic effect of the Pgp inhibitors Expression of proteins involved in apoptosis regulation

To study the effect of Pgp inhibition on cell proliferation we Expression of several proteins involved in the regulation of performed four replicate experiments measuring cell growth apoptosis, p53, Bcl-xL, Bcl-2, and Bax was assessed using in the three pairs of leukemia cell lines during single-drug immunoblotting (Figure 6). None of the cell lines expressed treatment with increasing doses of the resistance modulating the apoptosis transcription factor p53, which promotes agents. The growth of the KG1a/200 and K562/150 cells was apoptosis subsequent to DNA damage.41 On the other hand, inhibited in a dose-dependent manner by treatment with nan- expression of the pro-apoptosis Bax protein was moderate in omolar concentrations of either SDZ PSC 833, SDZ 280–446 all cell lines. Both the wild type and the MDR variant of KG1a or LY335979 (Figure 1), whereas the growth of the corre- cells expressed high levels of the anti-apoptotic proteins Bcl- sponding parental lines and both sensitive and resistant HL60 2 and Bcl-xL. Both K562 and both HL60 cell types expressed lines remained unaffected by equivalent treatment (Figure 2). high levels of Bcl-xL. Conversely, Bcl-2 was present at a low The half-maximal growth inhibitory doses for the responsive level in the K562 cells and basically absent in the HL60 cells. cells are listed in Table 1. Interestingly, there was no difference in the pattern of expression within each pair of MDR and wild-type cells. Thus, selection for drug resistance did not change the levels of the Pgp inhibition is associated with apoptosis induction apoptotic regulators studied in the KG1a, K562 and HL60 cells. Treatment with 1 ␮m SDZ PSC 833 for 48 h did not affect the expression level of these proteins either. Previously, we demonstrated by microscopy that exposure of The surface membrane protein APO1/Fas, which is an drug resistant KG1a cells to SDZ PSC833 resulted in chroma- important transducer of certain apoptotic signals,42 was meas- tin condensation and nuclear fragmentation,26 which are ured by flow cytometry and immunofluorescence detection. characteristic morphological features of apoptosis. In the From these analyses it was evident that APO1/Fas was present present study we measured DNA fragments resulting from in both HL60 cell types, and absent in both K562 cell types, apoptosis in cells exposed for 48 h to 1 ␮m of either SDZ PSC whereas differential expression of APO1/Fas was demon- 833, SDZ 280–446 or LY335979 using TUNEL assay and flow strated for the wild type and the MDR variant of KG1a cells cytometry. Cellular DNA content was measured simul- (Figure 7). Only the MDR variant was APO1/Fas positive, taneously. The results showed that the KG1a/200 and which might favor a cytotoxic effect of the resistance modu- K562/150 MDR variants were clearly apoptotic as demon- lators in these cells. strated by at least a two-fold increase in the FITC-fluorescence signal from dUTP-labeled DNA fragments compared to a neglible increase in the parental cells (Figure 3). Furthermore, striking changes in the DNA distribution of Discussion KG1a/200 cells occurred after treatment for 48 h with the modulators. In the control cultures the percentages of cells in Pgp belongs to the ATP binding cassette (ABC) family of trans- each cell cycle compartment were as follows: G1 phase 35%, porter molecules,43 which also include the MDR-associated S phase 35% and G2/M phase 30%. In the SDZ PSC 833- protein (MRP).44 These proteins contribute to MDR by their treated cultures the G1 phase population was reduced to 4%, functional activity as efflux pumps for a wide range of cyto- the S phase population to 6% and the G2/M phase population toxic agents. Furthermore, Pgp contributes to transmembrane increased to 62%, which indicates G2/M arrest. In addition, efflux of certain cytokines24,25 which may promote growth of cells of both higher (18%) and lower (10%) ploidy emerged, various leukemia cells45–47 and provide protection against the latter representing advanced stages of apoptosis with only apoptosis in vitro.48 Therefore, one possible consequence of remnants of the nuclei left in the cells. Similar results were Pgp inhibition could be reduction of growth stimuli available Selective cytotoxicity of P-glycoprotein inhibitory agents G Lehne et al 772

Figure 1 Growth inhibition curves for wild-type leukemia cells (KG1a/0, K562/0) and their resistant sublines (KG1a/200, K562/150) after exposure to increasing doses of SDZ PSC 833, SDZ 280–446 or LY335979. Both MDR variants responded to the three agents with signiﬁcant growth inhibition. Each point represent the mean of triplicates. The error bars represent 95% conﬁdence intervals.

to proliferating leukemia cells followed by apoptosis. In the iﬁed. The putative role for Pgp in the transmembrane transport present study we have demonstrated that Pgp overexpression of cytokines coincides with an autocrine growth regulation is associated with a growth inhibitory response to the Pgp involving a number of cytokines in cell lines derived from inhibitors SDZ PSC 833, SDZ 280–446 and LY335979 in the patients with AML. It has been demonstrated that leukemic MDR variants of KG1a and K562 leukemia cells. On the other myeloid blast cells generally express high levels of IL1 ␣, IL3, hand, the non-Pgp MDR variant of HL60 cells, which overex- and IL6 receptors,52 and that blast cells from AML patients presses MRP, was not receptive to similar growth inhibitory both express and produce a number of cytokines including effects. Thus, the cytotoxic effect of the modulators is associa- GM-CSF,53 IL-1,54 IL-6,55 IL-956 and IL-11.57 Furthermore, cell ted with Pgp in particular and not with MDR in general. proliferation may occur in autocrine loops based on endogen- The KG1a and K562 leukemia cells are poorly differentiated ously produced and extracellularly released growth fac- and share many characteristics with primitive hematopoietic tors.55,57 The mechanism behind this kind of cellular signal blast cells.49–50 Chaudhary and Roninson51 have proposed that transduction is unknown, but it is likely that any kind of inter- Pgp in hematopoietic stem cells may be involved in the export ference with the processing of vital growth factors, such as of a growth regulatory molecule which was hitherto not ident- transport inhibition by modulating agents, will have a detri- Selective cytotoxicity of P-glycoprotein inhibitory agents G Lehne et al 773 Table 1 The half-maximal growth inhibitory concentrations of SDZ PSC 833, SDZ 280–446 and LY335979 in resistant KG1a/200 and K562/150 leukemia cells

GI50 for

SDZ PSC 833 SDZ 280–446 LY335979

KG1a/200 312 (SE 41) 685 (SE 51) 66 (SE 1) K562/150 414 (SE 50) 578 (SE 54) 48 (SE 8)

The cells were cultured for 72 (K562) and 96 (KG1a) h with increasing concentrations of each modulator to obtain the concentration

required for 50% inhibition of cell growth (GI50). The values are means of four replicate experiments. Standard errors of the mean (SE) are given in parenthesis.

Figure 2 Growth inhibition curves for sensitive HL60/0 and resistant HL60/130 cells after exposure to increasing doses of SDZ PSC 833, SDZ 280–446, or LY335979. Growth inhibition did not occur in either of the cell types at the applied dosages. Each point represent the mean of triplicates. The error bars represent the 95% confidence intervals. mental effect on cell survival. Correspondingly, withdrawal of the supporting growth factor commonly leads to cell death by apoptosis within a few days in most factor-dependent cell lines, and the apoptotic process can be suppressed by new Figure 3 Overlay histograms showing the flow cytometric distri- addition of growth factors.58 bution of FITC-dUTP-labeled DNA nick ends in resistant KG1a/200 ␮m In the present study the TUNEL assay, the DNA distribution (a) and K562/150 (b) cells after treatment with 1 of SDZ PSC 833, SDZ 280–446 or LY335979 for 48 h in comparison with the corre- analyses and the microscopic images provided evidence that sponding wild-type cells. The column to the right lists the values of apoptosis contributes substantially to the selective cytotoxicity relative fluorescence (rF) which is the ratio between mean fluor- of the modulating agents studied. The MDR variant of escence values of treated (numerator) and untreated (denominator) KG1a/200 cells expressed the APO-1/Fas apoptosis trans- populations. The Y-axis represents the number of cells. Kolmogorov– Smirnov statistics revealed that dUTP fluorescence increased signifi- ducer, in contrast to the wild type, which suggests that this = = resistant line is more receptive to apoptosis induction than its cantly compared to non-treated cells for KG1a/200 (D 0.74, D 0.58 and D = 0.70 for SDZ PSC 833, SDZ 280–446 and LY335979, parent line. On the other hand, absence of APO-1/Fas does respectively) and K562/150 (D = 0.33, D = 0.33 and D = 0.54) cells. not prevent resistant K562/150 cells from undergoing The histograms presented are representative for three replicate experi- apoptosis, which indicates that apoptosis induced by SDZ PSC ments. Selective cytotoxicity of P-glycoprotein inhibitory agents G Lehne et al 774

Figure 4 Bivariate scatterplots presenting cell cycle distributions for apoptotic and viable KG1a/200 (a) and KG1a/0 (b) cells in 48 h regular cultures or cultures supplemented with 1 ␮m SDZ PSC 833, SDZ 280–446 or LY335979. Cellular DNA content is expressed as DNA-PI ﬂuor- escence (FL3) along the X-axis, and DNA strand breaks as dUTP-FITC labeling of DNA nick ends (FL1) along the Y-axis. Correspondingly, the horizontal histogram reveals the DNA ploidy (2N, 4N, 8N), and the vertical histogram depicts dUTP-FITC incorporation. The arrows demarcated as APO + and the horizontal cut-off lines in the dUTP-FITC histograms indicate apoptotic regions. Treatment of KG1a/200 cells with a modulator results in G2/M arrest, cytokinesis failure, polyploidization and apoptosis, whereas the parental KG1a/0 cells were unaffected. These data were representative for three replicate experiments.

833, SDZ 280–446 and LY335979 is independent of the APO- the present study. Inhibition of Pgp in these cells was also 1/Fas pathway. The BCR-ABL anti-apoptosis gene is a cyto- associated with cytokinesis failure as shown by the accumu- genetic hallmark of chronic myelogenous leukemia (CML),59 lation of large tetraploid populations and the emergence of and is constitutively expressed in the CML derived K562 cells, binucleated cells using ﬂow cytometry and microscopy, which accordingly, are highly resistant to apoptosis induction respectively. Thus, our study strongly indicates that over- by a variety of stimuli.60,61 Therefore, it is quite remarkable expression of Pgp is involved in acquisition of cytokinesis fail- that the MDR variant of K562 cells also undergoes apoptosis ure and apoptosis following exposure to Pgp inhibitors. Cells after exposure to nanomolar concentrations of the modulators with a low constitutive level of Pgp appear to be at less risk of studied. In addition, the anti-apoptotic regulators Bcl-2 and these events. Further studies are needed to identify the trigger Bcl-xL contribute to drug resistance of the MDR variants of mechanism(s) and apoptotic pathway(s). Currently, investi- both KG1a and K562 cells. Both Bcl-2 and Bcl-xL are fre- gations of these are being done using time course ﬂow cyto- quently coexpressed with Pgp in acute myeloblastic leukemia metric DNA analyses and cell sorting of cultures treated with cells and associated with autonomous growth in vitro and Pgp inhibitory agents. poor prognosis of leukemia.62,63 Development of hypersensitivity to cytotoxic drugs, local The apoptotic promoter p5364 was not expressed in any of anesthetics, narcotic analgesics and calcium antagonists dur- the cell lines studied, which is consistent with the total ing in vitro selection for drug resistance have previously been absence of p53 mRNA and protein expression previously reported as collateral sensitivity.70–72 Most of these com- reported for the wild types of KG1a,65 HL6066 and K56267 pounds are not substrates of Pgp, and the phenomenon has cells. Although loss of wild-type p53 function is usually asso- been associated with alteration of membrane biophysical ciated with resistance to apoptosis,68,69 the Pgp-rich variants properties rather than Pgp inhibition.70–73 Collateral sensitivity of KG1a and K562 were receptive to apoptosis induction in has been demonstrated for drug concentrations in the Selective cytotoxicity of P-glycoprotein inhibitory agents G Lehne et al 775

Figure 4 Continued

micromolar range.72,74 Conversely, the selective cytotoxicity to block MRP,75 and thus represents a less likely target for the of the modulators in this study appeared at considerably lower modulators studied. concentrations, which facilitates possible therapeutic exploi- For several reasons it appears that functional restriction of tation, and resulted specifically in cytokinesis failure and the Pgp efflux pump might be responsible for the particular apoptosis induction. The maximally tolerable plasma concen- phenomenon disclosed in this paper. First, the cytotoxicity tration of PSC 833 in cancer patients is 2.5–3 ␮m,19 which was conferred by drugs specifically targeted at Pgp interac- demonstrates that the concentrations applied in our study are tion. Second, the results were consistent irrespective of struc- clinically relevant. tural diversity of the Pgp inhibitory agents. Third, these agents The present paper discloses selective cytotoxicity of low were shown to induce functional inhibition of Pgp at compa- doses of the Pgp inhibitory agents SDZ PSC 833, SDZ 280– rable concentrations. Fourth, susceptibility to these agents 446 and LY335979. Previously, we have reported a similar required Pgp overexpression. Finally, expression of a specific ␮ effect of high-dose verapamil (GI50 11.6 m) and a 60-fold apoptotic regulatory protein did not seem to be the differential lower dose of SDZ PSC 833, which was associated with the factor between receptive and non-receptive cell lines. To MDR phenotype in leukemia cells but not in hepatoma cells.26 further prove the relationship between Pgp and the said In the present paper we demonstrate that increased MRP phenomenon experiments using homologue cell lines with expression is not a predisposing factor due to unrestrained different Pgp expression levels, cell lines transfected with the growth of MRP expressing HL60 leukemia cells in the pres- MDR1 gene, or cell lines depleted for Pgp by MDR1 antisense ence of the modulators, and efficient growth inhibition of the treatment are warranted. MDR variants of KGla and K562 leukemia cell lines induced In conclusion, our results demonstrate that low-dose treat- by LY335979 which does not interact with MRP. SDZ PSC ment of Pgp-rich leukemia cells with different Pgp inhibitory 833 and SDZ 280–446 appear to interact with both Pgp and agents results in growth inhibition and cell death by apoptosis. MRP, but the contribution of other transporters cannot be This finding represents a potential therapeutic option that is excluded from these experiments. Another MDR-associated currently being studied in vivo, and should be considered dur- ABC transporter, the multispecific organic anion transporter ing the ongoing development of strategies to overcome multi- (cMOAT), is only inefficiently inhibited by compounds known drug resistance. Selective cytotoxicity of P-glycoprotein inhibitory agents G Lehne et al 776

Figure 5 The microphotograph of resistant KG1a/200 leukemia cells treated with PSC 833 and stained with Papanicolau dyes demonstrates morphological features of cytokinesis failure and apoptosis. Examples of binucleated cells (A, B), cells with fragmented nuclei (C) and cells with condensed nuclei (D) are indexed.

Figure 7 Overlay histograms showing the flow cytometric distribution of fluorescence-labeled APO-1/Fas in wild types (KG1a/0, K562/0, HL60/0) and corresponding MDR variants (KG1a/200, K562/150, HL60/130) of human leukemia cells. The primary antibody was either DMS138 (a) or DMS140 (b). The column to the right lists the expression factor (EF) for APO-1/Fas which is the ratio between mean fluorescence values of populations stained with relevant (numerator) and irrelevant (denominator) MAbs. The Y-axis represents the number of cells. Histogram comparisons using Kolmogorov–Smir- nov statistics revealed that APO-1/Fas fluorescence differs significantly from the isotypic controls for KG1a/200 (D = 0.60 and 0.72 for DMS138 and DMS140, respectively), HL60/0 (D = 0.91 and 0.92) and HL60/130 (D = 0.93 and 0.92) cells. 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