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Research Article

α1-adrenergic drugs exhibit affinity to a thapsigargin-sensitivebinding site and interfere with the intracellular Ca2+

homeostasis in human erythroleukemia cells

Robert Fuchsa,⁎, Elisabeth Schraml a, b, Gerd Leitinger c, d, Ilse Letofsky-Papst e,Ingeborg Stelzer a, f, Helga Susanne Haasa, Konrad Schauenstein a, †, Anton Sadjaka

aInstitute of Pathophysiology and Immunology, Center of Molecular Medicine, Medical University of Graz, Heinrichstrasse 31A, 8010 Graz, AustriabInstitute of Applied Microbiology, University of Natural Resources and Applied Life Sciences Vienna, Muthgasse 18, 1190 Vienna, AustriacInstitute of Cell Biology, Histology and Embryology, Center ofMolecularMedicine, Medical University of Graz, Harrachgasse 21/7, 8010Graz, AustriadCenter for Medical Research, Core facility Ultrastructure Analysis, Medical University of Graz, Stiftingtalstrasse 24, 8010 Graz, AustriaeResearch Institute for Electron Microscopy, Graz University of Technology, Steyrergasse 17, 8010 Graz, AustriafClinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria

A R T I C L E I N F O R M A T I O N

⁎ Corresponding author. Fax: +43 316 380 964E-mail address: robert.fuchs@medunigraz.at

† Deceased on May 22, 2007.

0014-4827/$ – see front matter © 2011 Elseviedoi:10.1016/j.yexcr.2011.08.003

A B S T R A C T

Article Chronology:

Received 1 July 2011Accepted 2 August 2011

Available online 9 August 2011

Even though the erythroleukemia cell lines K562 and HEL do not express α1-adrenoceptors, someα1-adrenergic drugs influence both survival and differentiation of these cell lines. Since Ca2+ isclosely related to cellular homeostasis, we examined the capacity of α1-adrenergic drugs to

modulate the intracellular Ca2+ content in K562 cells. Because of morphological alterations ofmitochondria following α1-adrenergic agonist treatment, we also scrutinized mitochondrialfunctions. In order to visualize the non-adrenoceptor binding site(s) of α1-adrenergic drugs inerythroleukemia cells, we evaluated the application of the fluorescent α1-adrenergic antagonistBODIPY® FL-Prazosin. We discovered that theα1-adrenergic agonists naphazoline, oxymetazolineand also theα1-adrenergic antagonist benoxathian are able to raise the intracellular Ca2+-contentin K562 cells. Furthermore, we demonstrate that naphazoline treatment induces ROS-formation aswell as an increase in Δψm in K562 cells. Using BODIPY® FL-Prazosin we were able to visualize thenon-adrenoceptor binding site(s) of α1-adrenergic drugs in erythroleukemia cells. Interestingly,the SERCA-inhibitor thapsigargin appears to interfere with the binding of BODIPY® FL-Prazosin.Our data suggest that the effects of α1-adrenergic drugs on erythroleukemia cells are mediated by

a thapsigargin sensitive binding site, which controls the fate of erythroleukemia cells towardsdifferentiation, senescence and cell death through modulation of intracellular Ca2+.

© 2011 Elsevier Inc. All rights reserved.

Keywords:

Erythroleukemia cellsα1-adrenergic drugsMitochondriaCalciumReactive oxygen species

Introduction

In the preceding paper we presented thatα1-adrenergic drugs canmodulate cell death, differentiation, autophagy as well as the

0.(R. Fuchs).

r Inc. All rights reserved.

growth behavior of the human erythroleukemia cell lines K562and HEL [1]. Since these cell lines do not express α1-adrenergicreceptors [1], the physiologic relevance of this observation regardingthe interactionof thesympathetic nervous systemanderythropoiesis

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[2,3] are unclear, as yet. The non-adrenoceptor binding site(s) ofα1-adrenergic drugs in erythroleukemia cells has/have not beenidentified, so far. Nevertheless, the observed effects ofα1-adrenergicdrugs on erythroleukemia cells are worth being investigated infurther detail. First, the binding capacity of norepinephrine towardstheunknowntarget has tobe evaluated. ThegrouparoundLivingstonhas shown that after application of high norepinephrine doses in ratsthe in vitro growth of erythroid colonies is inhibited [3]. It cannot beexcluded that norepinephrine shows affinity to the unknown targetrecognized by synthetic α1-adrenergic drugs at least in high doses.Furthermore,α1-adrenergic drugs could be valuable tools for furthercharacterization of differentiation processes and regulation of celldeath in erythroleukemia cells and also in physiologic erythroidprogenitor cells. The most important reason, however, is that wecould show that α1-adrenergic antagonists are able to inducedifferentiation and apoptosis in leukemia cells [1]. Therefore, theidentification of the unknown binding site might disclose a newtarget for the treatment of leukemia and other types of cancer. Thechemical structures of α1-adrenocepter modulators could be thebasis for the development of new cancer drugs. In addition,knowledge about the identity of non-adrenoceptor-targets of α1-adrenergic drugs couldbehelpful to designnewα1-adrenergic drugswith high specificity for adrenoceptors but withminor affinity to thenon-adrenoceptor target.

In the preceding paper we showed that the naphazoline(naph)-induced aggregation of K562 cells can be mimicked,respectively enhanced, by chelation of Ca2+ with the Ca2+

selective chelator BAPTA [1]. Therefore, we here analyzed thepotential of different α1-adrenergic drugs to influence the cellularCa2+-content in K562 cells. Cellular Ca2+-homeostasis is closelylinked to pathologic effects such as endoplasmatic reticulum (ER-)stress, autophagy and apoptosis [4–6]. Moreover, Ca2+-ions playan important role as second messenger molecules in the signallingcascade of erythropoietin and, generally, in the process oferythroid differentiation [7,8]. Ca2+-ions are also essential forthe function of mitochondria [5]. Our data presented in theprevious paper show that themorphology of mitochondria in naphtreated cells is altered in comparison to untreated cells, with ahigher number of mitochondrial granules in naph treated cellsthan in untreated cells [1]. It is well known that metal ions such asCa2+, iron or copper accumulate in mitochondria in pathologicconditions or diseases like mitochondrial dysfunction and ROSgeneration (calcium, [5]), sideroplastic anemia (iron, [9]), orWilson's disease (copper, [10]). In order to define the role ofmitochondria in the naph-induced toxicology in K562 cells, weanalyzed the molecular composition of mitochondrial granules innaph treated cells by means of analytical electron microscopy.Because previous studies showed that in vitro as well as in vivotreatment of hematopoietic cells with norepinephrine respectivelyadrenergic agonists resulted in enhanced cellular ROS levels [11,12],we further characterized mitochondrial function by measuring themitochondrial membrane potential and cellular generation of reac-tive oxygen species (ROS).

In order to visualize and to localize the non-adrenoceptorbinding site of α1-adrenergic drugs in erythroleukemia cells weused the fluorescentα1-adrenergic receptor antagonist BODIPY®FL-Prazosin, designated by Daly et al. as quinazoline piperazine-BODIPY(QAPB) [13,14]. QAPB is a quinazoline based α1-adrenoceptorantagonist like prazosin, but the furan-residue of prazosin issubstituted by a fluorescent BODIPY®-residue [13].

We demonstrate here that α1-adrenergic drugs are able tomodulate the intracellular Ca2+ content in erythroleukemia cells.Furthermore, we show that the toxicology of naph in K562 cells islinked to the generation of reactive oxygen species and mitochon-drial dysfunction. Finally, we present a successful stainingprocedure of the non-adrenoceptor target recognized by α1-adrenergic drugs in erythroleukemia cells, which could be thebasis for further identification of the unknown target. As firstindication for the identity of the unknown binding site we coulddefine that the unknown target exhibits affinity to the SERCA-inhibitor thapsigargin.

Materials and methods

Cultivation of leukemia cells with adrenergic drugs

Human erythroleukemia cell lines K562 and HEL cells werecultivated under the same conditions and with the same drugsas described in the previous paper [1]. The adrenergic agonistsnaphazoline HCl (naph, α1) and oxymetazoline HCl (α1/partialα2)—each dissolved in cell culture medium—were added indifferent concentrations to cultures of erythroleukemia cells. Theadrenergic antagonists benoxathian HCl (benox, α1), prazosin HCl(α1) or yohimbine HCl (α2) were added to cell cultures asaqueous solutions. All adrenergic drugs were obtained from Sigma(St. Louis, MO/USA), except oxymetazoline which was purchasedfrom ICN Biomedicals (Aurora, OH/USA).

Analytical transmission electron microscopy

To dissect the elemental composition of distinct observed ultra-structures in erythroleukemia cells, analytical transmission electronmicroscopy (TEM)was performed on a Philips CM 20 TEMequippedwith a Gatan imaging filter. Elemental analyses were conducted bymeans of energy filtering TEM (EFTEM) and energy dispersive X-rayspectroscopy (EDXS) as described in detail by Pabst et al. [15]. EDXSenables the analysis of the qualitative and the relative quantitativeelemental composition of illuminated specimen areas by assessingthe energy and the intensity of emitted X-rays [15]. For elementalanalysis ultra-thin sections were collected on copper or nickel-gridsand were analyzed without further contrasting.

Acridine orange staining of K562 cells

In order to detect acidification, respectively, to visualize lysosomesin K562 cells, cells were stained with acridine orange as describedin the preceding paper [1].

Analysis of reactive oxygen species (ROS) and the mitochondrialmembrane potential (Δψm) in K562 cells

For detection of ROS generation, naph treated K562 cells wereharvested and re-suspended in a staining buffer on the basis of CMF-PBS containing 10 μM 2′7-dichlorodihydrofluorescein-diacetate(DCF, Sigma). To assess autofluorescence, cells of the same probewere incubated with buffer containing DMSO, which was used assolvent for preparation of the DCF-stock. Cells were incubated in awater bath at 37 °C for 15 min in the dark, washed once, re-suspended in CMF buffer and analyzed with a FACScan flow

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cytometer immediately. Untilmeasurement stained cellswere storedprotected from light in an ice box. As positive control themitochondrial ROS-generator menadione (Sigma) was used [16].

In order to protect cells against enhanced ROS-generation,K562 cell cultures were supplemented with the antioxidant drugN-acetyl-cysteine (NAC, Sigma).

For analysis of the mitochondrial membrane potential (Δψm)of K562 cells the JC-1 Mitochondrial Membrane Potential AssayKit of Cayman Europe (Tallinn, Estonia) was used. Cells wereanalyzed by flow cytometry according to the instructions of theproducer. As control, untreated or naph treated cells wereincubated with 10 μM of the depolarizing ionophore carbonylcyanide-4-trifluoromethoxyphenylhyrazone (FCCP, Sigma) [17].

Visualization of the non-adrenoceptor binding site of α1-adrenergic drugs in erythroleukemia cells using BODIBY® FL-Prazosin

To characterize the unspecific binding of adrenergic drugs inerythroleukemia cells, cells were analyzed by flow cytometry andfluorescence microscopy using the fluorescent α1-adrenergicantagonist BODIPY® FL-Prazosin (QAPB, Invitrogen/MolecularProbes, Eugene, OR/USA). For ligand binding assays erythroleukemiacells were harvested, washed once with RPMI-1640 and 2× E5 cellswere incubated in a total volume of 400 μl RPMI-1640with/withoutaddition of a test ligand for 1 h at 37 °C in a water bath. Followingpre-incubation with the test ligand, cells were loaded with 100 nMQAPB and incubated, protected from light, for further 30 min at37 °C. Subsequent to incubation, cells were washed once with icecoldphosphatebuffer, re-suspended inbuffer andwereplacedon iceunder light protection until flow cytometry or fluorescence micros-copy inspection.

QAPB-derived fluorescence of the cells was measured at aFACScan flow cytometer at the FL-1 channel. To estimateautofluorescence of cells, unstained cells were analyzed prior toQAPB stained cells. To assess the specificity of QAPB to recognizethe prazosin binding site in erythroleukemia cells, prazosin andL-(−) norepinephrine (+)-bitartrate salt monohydrate (NE, Sigma)were used as control ligands. NE-solutionswere prepared separatelyfor each experiment, immediately before addition to the cellsuspensions. In the flow cytometry assays, cells were further stainedwith 7-amino-actinomycin D (7-AAD, Sigma) to discriminate deadcells.

Measurement of cellular calcium content

The relative cytoplasmic calcium content of K562 cells was assessedby flow cytometry as a time function, using Calcium Green™-1, AM(CG, Invitrogen/Molecular Probes) according to protocols providedby Piwocka et al. [18] and Silei et al. [19] with several modifications.Shortly, cells were harvested, washed once with RPMI-1640withoutphenol red (PAA) and re-suspended in the same medium, 0.5×E6cells/ml, before loading with 1 μM CG for 20 min at 21 °C.Subsequently, cells were washed twice with medium. After loadingof the cells, the relative basal cytosolic Ca2+-contentwas determinedfollowing a 25 min recovery period at 37 °C by measuring cellularCG-fluorescence at the FL-1 channel of a FACScan for 51 s. In order toanalyze time kinetics of the relative cytoplasmic calcium concentra-tion, all measurements were performed at 37 °C. This was achievedbyusing a sample holder of a FACSvantage cell sorter (BD),which can

be tempered by a circulatingwater bath. After estimating the steadystate fluorescence of CG-loaded cells, cells were stimulated withadrenergic agents or other ligands. Then, changes of cellularfluorescence were analyzed as a function of time. Ionomycin(2 μM, Tocris Bioscience, Bristol, UK) and thapsigargin (5 μM, Tocris)were used to establish the method and as positive controls. Relativechanges in cytoplasmic calcium content were calculated as ΔF/F0=100((F−F0)/F0), where F0 is themedian fluorescence of steadystate cells and F the median of fluorescence of cells of continuousperiods of 4 s [18].

Statistics

Results are expressed as mean values plus or minus standarddeviation. Data were analyzed by the unpaired Student's t testusing Sigma Plot 11.0. p-Values equal or less than 0.05 wereconsidered as significant.

Results

α1-adrenergic agonist-induced cell death of K562 cells is triggeredby reactive oxygen species and can be influenced by modulatingintra-, and extracellular Ca2+

To investigate the role of ROS in the toxicology of naph inerythroleukemia cells, we measured the capacity of treated anduntreated K562 cells to oxidize DCF. Following 24 h cultivation ofK562 cells, we could not observe any difference in the oxidizingcapacity between naph treated cells and untreated controls. At48 h, the pro-oxidative activity of 100 μM naph treated K562 cellsis enhanced in comparison to untreated cells (Fig. 1A). In contrast,200 μM naph treated cells show an overall lower pro-oxidativeactivity than the untreated control after 48 h (Fig. 1A). Backgroundfluorescence of K562 cells is not influenced followingnaph treatment,as determined by analyzing unstained cells (not shown). In order tounderpin the participation of ROS in the naph-induced effects, K562cultures were co-treated with 200 μM naph and the commonly usedantioxidant molecule n-acetyl-cysteine (NAC). 5 mM NAC protectsK562 cells effectively against the toxic effect of naph (Fig. 1B),whereas at higher concentrations (10–15 mM) NAC inhibits thegrowth of K562 cells by itself (Fig. 1B).

Prompted by the result that the naph-induced cellularaggregation can be mimicked by chelation of extracellularcalcium [1], we raised the Ca2+-concentration in the medium byadding CaCl2 (2–5 mM) together with 200 μM naph. The supple-mentation with CaCl2 does not influence the growth of the K562cells per se, but dose-dependently and significantly protects K562cells against the toxicity of naph, albeit the protective mechanismis just slightly pronounced (Fig. 1C). In addition to this growth-restoring effect, the aggregation of the cells is also abrogatedunder raised Ca2+-concentrations (not shown). We furtherexplored the effect of ionomycin on naph treated K562 cells,which elevates intracellular Ca2+ by permitting Ca2+-influxthrough the plasmalemma. In combination with naph we couldsee that even though ionomycin does not further influence theproliferation capacity of naph (200 μM) treated K562 cells, itsignificantly maintains the viability of the cells (Fig. 1D). LikeCaCl2, ionomycin also efficiently abolishes the naph-induced cellaggregation (not shown).

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Fig. 1 – α1-adrenergic agonist-induced necrotic cell death is triggered by reactive oxygen species (ROS) and Ca2+. A: ROS-analysis inK562 cells subsequent to incubation with naphazoline (naph). Data are presented as fold change of mean fluorescence at FL-1following incubation of K562 cells with 2′7-dichlorodihydrofluorescein-diacetate (DCF) in comparison to untreated controls.Whereas 100 μM naph boost the generation of ROS, 200 μM of the drug lowers the overall pro-oxidative capacity of K562 cells incomparison to untreated cells. 24 h: n=4, 48 h: n=3, *p<0.05 according to unpaired t-test. B: n-acetyl-cysteine (NAC) neutralizesthe toxic effect of 72 h naph treatment on K562 cells. Data are presented as the relative number of living cells in comparison tountreated controls (=100%). n=3, ***p<0.001 versus naph-treated cells, ###p<0.001 versus untreated control, according tounpaired t-test. C/D: K562 cells can be protected against naphazoline-induced cell stress by addition of CaCl2 or ionomycin to theculture medium. Whereas addition of Ca2+ restores proliferation (C) to a small extend in 72 h naphazoline (naph) treated cultures,ionomycin does not interfere with the naph-induced growth inhibition, but maintains the viability of cells (D) in the presence ofnaph. C/D: n=4, *p<0.05, **p<0.01, ***p<0.001 according to unpaired t-test. E: Naphazoline-induced acidification in K562 cells isreversible by treatment with NAC. Acridine orange (AO) staining was performed in order to assess acidification respectivelylysosomal integrity of naph treated cells. K562 cells were treated 48 h with naph (200 μM) with/without addition of 5 mM NAC,5 mM CaCl2 or 5 μM ionomycin (iono). The fluorescence figures are overlays, generated from figures of the nuclear staining of AOand the staining of acidic organelles. Naph treatment of K562 cells results in acidification of the cells, reversible by NAC but onlyslightly by CaCl2 and ionomycin.

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Since we demonstrated in the preceding paper by means ofacridine orange (AO) staining [1] that naph induces acidification inK562 cells, we repeated the AO staining with cells treated withnaph and NAC, respectively, CaCl2 and ionomycin. Acridine orange

staining of K562 cells treated with naph and NAC suggests thatROS-generation and cellular acidification are linked together, sinceNAC (5 mM) efficiently abrogates acidification of the cells (Fig. 1E).Manipulation of extra- and intracellular Ca2+-concentration with

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CaCl2 (5 mM) or ionomycin (5 μM) only slightly restores acidifi-cation in naph treated cells (Fig. 1E).

α1-adrenergic drugs induce a raise of intracellular Ca2+ in K562cells

Calcium-assays demonstrated that naph and oxymetazoline mod-erately elevate the cytoplasmic calciumcontent in K562 cells (Fig. 2I/IV). Although the height of the amplitude is similar for naph andoxymetazoline, the oxymetazoline-induced Ca2+-raise persists fora longer period of time than observed following naph treatment(Fig. 2I/IV). Interestingly, the adrenergic antagonist benoxathian(benox) is also able to elevate cytoplasmic Ca2+ in K562 cells, evenwith higher potency than observed with naph or oxymetazoline(Fig. 2II). Benox immediately stimulates a raise of intracellular Ca2+

after addition of the drug to the cells, before the calcium levelis declining and stays constant at an elevated level. The maxi-mum height of the Ca2+-peak in response to benox treatment inK562 cells is similar to the peak observed following treatmentwith thapsigargin (Fig. 2II)—with the exception that thapsigargininduces a second peak of Ca2+-elevation. We interpret this secondpeak as store operated calcium entry (SOCE) [20]. Prazosin doesnot influence the cellular calcium content in K562 cells (Fig. 2V/VI).Overall, the calcium-elevation stimulated by benox or thapsigargindoes not reach the level of ionomycin (2 μM), which induces amean ΔF/F0 value of 70.78 (n=2) directly after stimulation of the

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Fig. 2 – Adrenergic drugs induce a raise of intracellular Ca2+ in Kstimulated with adrenergic drugs. Relative changes of cellular Caflow cytometry as a time function. Subsequent estimating the cellby the arrows) with naphazoline (Naph, [200 μM]), oxymetazoline (O[40 μM]), yohimbine (Yoh, [40 μM])or thapsigargin (Thapsi, [5 μM]). Iwith twodrugs (Benox+Thapsi, Benox+Naph)orpre-treatedwith aPrazo+Benox, Yoh+Naph) and stimulated subsequently with thn=12, Prazo: n=5, Thapsi: n=11, Benox+Thapsi: n=3, Prazo+TBenox: n=6, Yoh+Naph: n=4.

cells. This indicates that benox- and thapsigargin-induced calciumresponses do not reach the upper limit of detection in the assay. Toexplore whether the effects of benox and thapsigargin interferewith each other, cells were stimulated simultaneously with bothdrugs. The results of these experiments showed that Ca2+ iselevated similar as observed in cells treated with benox orthapsigargin alone, but following the primary response the cellularcalcium content immediately decreases (Fig. 2II). On the contrary,pre-treatment of K562 cells with prazosin does not influence thecalcium elevation induced by thapsigargin (Fig. 2V).

Treatment of cells with both benox and naph also does notinfluence the height of the amplitude of Ca2+-elevation incomparison to cells treated with benox alone, but naph abrogatesthe benox-induced prolonged elevation of calcium levels in K562cells (Fig. 2III). A similar result can be obtained when cells are pre-treatedwithprazosin and then stimulatedwithbenox (Fig. 2III). Pre-treatment of cells with prazosin or yohimbine does not influencecellular calcium levels in response to naph-treatment (Fig. 2VI).

Naphazoline treatment induces a dose dependent increase of themitochondrial membrane potential (Δψm) in K562 cells

Since we observed that mitochondria of naph treated K562 cellsexhibit morphological alterations [1], and because mitochondriaare a major source of cellular ROS, we analyzed the mitochondrialmembrane potential (Δψm).

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562 cells. K562 cells were loaded with Calcium GreenTM and2+-levels as indicated by the ΔF/F0 value were monitored byular steady state fluorescence, cells were stimulated (indicatedxy, [200 μM]), benoxathian (Benox, [40 μM]), prazosin (Prazo,n combinationexperiments cellswere stimulated simultaneouslyα-adrenergic antagonist for 5 min (Prazo+Thapsi, Prazo+Naph,e second drug. Vector: n=19, Naph: n=8, Oxy: n=4, Benox:hapsi: n=4, Naph+Benox: n=5, Prazo+Naph: n=5, Prazo+

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Analysis of Δψm by means of the potentiometric dye JC-1[17,21,22] showed that naph treatment of K562 cells results inhyperpolarization of mitochondria as indicated by a dose depen-dent increase of red fluorescence emitted by JC-1 stained cells(Fig. 3). Simultaneous administration of JC-1 and the uncouplingreagent FCCP [10 μM] to K562 cells reverses the fluorescencesignals of naph treated cells (Fig. 3C). Unexpectedly, 10 μM FCCPfailed to decrease the median Fl-2 signal emitted by untreatedK562 cells (Fig. 3C). However, the reversible increase of the FL-2

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signal due to naph treatment and additional analysis of cells byfluorescence microscopy (Fig. 3F) confirms the increase of themitochondrial membrane potential in naph treated K562 cells.

Mitochondria of naph treated cells exhibit granules of nonmetallic nature

As described in the previous paper, mitochondria displayed anoverall more pronounced matrix density in naph treated K562

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Fig. 4 – Mitochondria of naphazoline treated K562 cells exhibit granules of non-metallic nature. Mitochondria of naphazolinetreated K562 cells (picture within the graph) exhibit electron dense dots (arrows), which elemental composition was dissected byEDXS. A representative EDXS graph of one electron dense dot is shown. Each peak in the graph refers to a specific element, asdesignated by the respective element symbol.

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cells and specific granules could be observed in comparison tountreated cells [1]. In order to explain the toxicology of naph onerythroleukemia cells, we analyzed the elemental composition ofthese granules. Analytical ELMI of mitochondrial granules revealedthat molecules consisting of carbon, oxygen, nitrogen and siliconform these structures (Fig. 4). Furthermore, molecules in thegranules exhibit high affinity to the fixative osmium. No evidencecould be obtained for the presence of metal ions like calcium, ironor copper. In the lipofuscin granules we also did not find any signsof metal ions, which normally comprise less than 2% of thelipofuscin mass, by EDXS analysis [23].

The fluorescent α1-adrenergic antagonist BODIPY® FL-Prazosin(QAPB) binds to an intracellular, thapsigargin-sensitive bindingsite in human erythroleukemia cells

To localize the potential non-adrenoceptor binding site(s) ofadrenergic drugs in human erythroleukemia cells, the fluorescentα1-adrenergic antagonist QAPB was used. In comparison to HELcells, K562 cells show a significant (p<0.001) higher capacity tobind QAPB (Fig. 5A). The fluorescence signal is not influenced bytreatment with 0.2% sodium azid in both cell lines, indicating no

Fig. 3 – Naphazoline treatment increases the mitochondrial membrfollowing naphazoline (naph)-treatment were examined by meansmitochondria and reversibly changes color from green to red as thwith naph and stained with JC-1 reagent. Emitted fluorescence of ththe FL-1 (green) respectively the FL-2 (red) channel. A: As assessedconcentrations induce cell death in K562 cells indicated by the appe7-AAD histogram. Therefore, solely cells lying in the region R1 (=ncells results in a dose-dependent decrease of the green fluorescenceindicating an increase of the Δψm in K562 cells due to naph treatmuncoupling reagent FCCP parallel to JC-1. Parallel treatment with 10treated cells but not of untreated cells. D/E: Statistics of fluorescencein a dose dependent increase of themedian of the emitted cellular refluorescence of JC-1 stained cells. Control=100%, n=3. E: The FL-2Control=100%, n=3. F: Analysis of cells by fluorescence microscopof K562 cells with naph increases the red fluorescence signal of JC-

ATP-dependent drug removal (not shown). In K562 cells granularfluorescence signals can be detected in the cytoplasm, suggestingintracellular binding of QAPB in the absence of α1-adrenoceptors(Fig. 5E). In the HEL cell line this granular staining is lesspronounced than in the K562 cell line (Fig. 5E). When K562 cellsare pre-treated with prazosin, the QAPB fluorescence signal showsa concentration-dependent characteristic. With 10 μM prazosinthe fluorescence signal is significantly (p<0.05) enhanced incomparison to control (Fig. 5B). Further increasing prazosinconcentrations dose-dependently suppress the fluorescence sig-nal. In the HEL cell line the fluorescence signal is not abrogated byprazosin but is enhanced over the whole range of testedconcentrations. Nevertheless, there is a clear trend that thefluorescence signal is declining dose-dependently with concen-trations higher than 25 μM (Fig. 5B). By microscopic analysis of10 μM prazosin treated cells we observed a perinuclear fluores-cence signal, suggesting that prazosin treatment leads to aggre-gation of a distinctive sub cellular component. 1.5 h treatment ofK562 cells further reduces the relative cell size of K562 cells in adose-dependent manner, without induction of cell death. This sizeeffect is reversible by sodium azid and is exclusively observed inthe K562 cell line (Fig. 5F). Benox causes a stronger extinction of

ane potential (Δψm) of K562 cells. Changes in Δψmof K562 cellsof the potentiometric dye JC-1, which selectively enters intoe membrane potential increases. K562 cells were treated 48 he cells was evaluated in an endpoint assay by flow cytometry atby (separate) staining with 7-AAD, high (200 μM) napharance of a pronounced peak of 7-AAD positive cells (red) in theegative for 7-AAD) were analyzed. B: Naph treatment of K562signal and a concomitant increase of the red fluorescence signal,ent. C: In order to control the assay, cells were treated with theμMFCCP successfully reversed the fluorescence pattern of naphsignals emitted by naph treated cells. D: Naph treatment resultsd fluorescence and a concomitant decrease of the emitted green/FL-1-ratio of K562 cells is increasing with naph treatment.y confirmed the results obtained by flow cytometry. Treatment1 stained K562 cells.

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Fig. 5 – The fluorescent α1-adrenergic antagonist QAPB binds to an intracellular, thapsigargin-sensitive binding site in humanerythroleukemia cells. K562- and HEL-cells were incubated 1 h with/without adrenergic drugs or other ligands and were loadedwith QAPB. Fluorescence of the cells was assessed by flow cytometry and fluorescence microscopy. A: K562 cells exhibit a highlysignificant (*p<0.001) stronger capacity to bind QAPB in comparison to HEL cells. K562: n=17, HEL: n=14. B: QAPB-bindingkinetics of erythroleukemia cells following pre-incubation with the α1-adrenergic antagonists prazosin or benoxathian (benox).K562: prazosin: n=4, benox: n=3, HEL: prazosin: n=3, benox: n=3. C: The Ca-ATPase-inhibitor thapsigargin extincts QAPBderived fluorescence of human erythroleukemia cells. K562: n=4, HEL: n=3. D: QAPB binding kinetics of erythroleukemia cellsfollowing pre-incubation with the α1-adrenergic agonists naphazoline (naph) and oxymetazoline (oxy) in comparison tonorepinephrine (NE). HEL: NE: n=3, naph: n=3, oxy: n=3, K562: NE: n=3, naph: n=4, oxy: n=3. E: QAPB-loadederythroleukemia cells exhibit granular fluorescence signals located in the cytoplasm, much stronger pronounced in K562 cells thanin HEL cells. Treatment of K562 cells with 10 μM prazosin and QAPB results in the formation of strong perinuclear fluorescencesignals. Original magnification: 630×. F: 1.5 h treatment with prazo results in a decrease of cell size (FSC-Height) in K562 cells.

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QAPB-derived fluorescence than prazosin in the K562 cells butexhibits no or just a weak effect in the HEL cell line (Fig. 5B).

In comparison to antagonists, the adrenergic agonistsnaph displace QAPB with minor potency. Whereas naph showssimilar potency to block QAPB in K562 and HEL cells, the moreeffective agonist oxymetazoline eliminates QAPB fluorescencestronger in the K562 cell line than in the HEL cells (Fig. 5D). Thephysiologic adrenergic agonist norepinephrine does not signifi-cantly influence QAPB-derived fluorescence in both tested celllines (Fig. 5D).

Sincewe observed that adrenergic drugs can elevate intracellularCa2+ similar as thapsigargin, we examined the capacity ofthapsigargin to replace the binding of QAPB in erythroleukemiacells. Indeed, QAPB derived fluorescence can be eliminated bythapsigargin in K562 cells, even in much lower concentrations thanα1-adrenergic drugs (Fig. 5C). Higher thapsigargin concentrations(≥ 8.5 μM) induce a dose-dependent increase of dead (7-AAD+)cells (not shown). In the HEL cell line the thapsigargin fluorescencesuppression-curve shows a similar characteristic as observed withprazosin (Fig. 5C).

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Fig. 6 – Final summary: Differentiation, senescence and cell death of human erythroleukemia cells are modulated by α1-adrenergicagents through a non adrenoceptor, thapsigargin sensitive binding site. Themost important effects of several α1-adrenergic drugs usedin the previous paper [1] and in the current study on K562 cells are shown. Even though K562 cells do not express α1-adrenoceptors,drugs originally designed to either blockor to activateα1-adrenoceptors influence cellularhomeostasis of humanerythroleukemia cellsthrough a, as yet, not identified binding site, which shows affinity to the SERCA-inhibitor thapsigargin. The unknownbinding site can bevisualized bymeansof the fluorescent adrenergic antagonist QAPB.α1-agonists inhibit differentiation and induce signs of senescence asthe generation of reactive oxygen species (ROS), mitochondrial dysfunction as well as accumulation of lipofuscin and subsequentnecrotic cell death. In contrast,α1-antagonists induce differentiation and apoptosis. As yet, it is not clearwhetherα1-antagonists inducedifferentiation and subsequent apoptosis or induction of differentiation and apoptosis are independent events. α1-agonists are able toattenuate theactionsofα1-antagonistsonhumanerythroleukemia cells includingbothdifferentiationandapoptosis. Ca2+-ionsseemtoplay an essential role in the decision of differentiation in K562 cells, since treatment of K562 cells with benoxathian, which induceshemoglobinization, evokes an intracellular Ca2+-signal, whereas prazosin, which induces endomitosis in K562 cells, is not able toinfluence intracellular Ca2+. The role of Ca2+ in the naphazoline-induced toxicology on K562 cells remains to be defined.

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Discussion

α1-adrenergic agonists induce signs of cellular senescence inK562 cells

In the current and the preceding paper we determined thegeneration of reactive oxygen species, proven by the existence ofenlarged lipofuscin granules in the cytoplasm [1] and ROS-measurements, as underlying mechanism of naph-induced celldeath in K562 cells. According to Terman et al. and other authors,lipofuscin originates from autophagocytized cellular components

(in special mitochondria) that have become oxidized outside orinside the lysosomal compartment [23–26]. Lipofuscin taken up bylysosomes is supposed to be reactive andmight lead to a rupture oflysosomal membranes by ROS-generation through metal ioncatalyzed redox-reactions [23,26]. Lysosomal membrane ruptureresults in the release of lysosomal enzymes into the cytoplasm,initiating self digestion of the cell [27]. The ghost-like phenotype ofnaph treated cells, as reported in the preceding paper [1], as well asthe observed acidification of the cells support the hypothesis thatthis scenario occurs in K562 cells in reaction to naph treatment.Nevertheless, it cannot be excluded, so far, that the diffuse orangecolour of cells following acridine orange staining results from

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general cellular acidification due to dysregulation of the acid–basebalance of the cells. The antioxidant NAC did not only suppressnaph-induced acidification of the cells, but also restored thegrowth of K562 cells in the presence of naph. These protectiveeffects of NAC prove the relationship between naph-inducedtoxicology and ROS generation in K562 cells.

ROS generation and lipofuscin formation are seen as majorhallmarks of postmitotic and senescent cells and are, according tothe mitochondrial–lysosomal theory of aging, important partici-pants in the cellular aging process [23–26]. Accumulation oflipofuscin diminishes lysosomal degradative capacity of cells byexhausting the pool of lysosomal enzymes, which hampersmitochondrial recycling by autophagy [26]. The consequences oflipofuscin formation in the cell are both lysosomal and mitochon-drial dysfunction, which paves theway towards cell death [23–26].The naph-induced effects fit well into the picture of this theory,since naph treated cells show large lipofuscin granules, enhancedautophagy as well as functionally altered mitochondria.

Mitochondria of naph treated cells exhibited electron densegranules in their matrix, which may consist of accumulatedproteins and/or lipids [1,28]. We have drawn this conclusionsince we could not obtain evidence for the presence of calcium,iron or copper in the granules [5,9,10]. To our surprise, K562 cellsshowed decreased ability to oxidize DCF after 48 h naph (200 μM)treatment. By examining the Δψm by means of JC-1 reagent, wecould demonstrate that naph induces mitochondrial hyperpolar-ization in K562 cells, which indicates mitochondrial dysfunction[21,22]. Our observation that treatment with high (200 μM) naphconcentrations results in a diminishedpro-oxidative capacity of K562cells is in linewith the studyof Schauen et al., whichhas revealed thatrespiratory chain deficiency results in reduced ROS generation inHeLa cells [29]. The occurrence of mitochondrial dysfunction is alsosupported by the observed morphological alterations of mitochon-dria, the disability of hemoglobinization in naph treated cells and thefact that apoptosis is not involved in naph-induced cell death [1].Apoptosis is a cellular process, which is generally known to bedependent on ATP generation by mitochondria [30].

In the absence of a functional NADPH-oxidase [31], the mito-chondria are the main source of ROS in K562 cells. Therefore andbecause of the observed functional and morphological alterations,the mitochondria are supposed to be the primary targets of naph.Nevertheless, the role of the ER in the naph-induced toxic reactionin K562 cells still needs to be investigated. For instance, Mao et al.revealed in the pheochromocytoma cell line PC-12 that norepi-nephrine induces ER-stress, associated with ROS-generation andinhibition of the Akt-kinase, which is an important factor in theinduction of differentiation in erythroid cells [32,33].

α1-adrenergic drugs modulate intracellular Ca2+—a key factorof cellular survival and differentiation—in erythroleukemia cells

In our current study we could confirm that the toxic effect of naphin K562 cells is linked to cellular calcium homeostasis. Thisobservation strengthens the hypothesis that mitochondria and/orthe ER are the primary targets of naph. Both organelles can storeCa2+ and are in close communication through Ca2+-ions [34].Dysregulation of Ca2+-homeostasis is discussed extensively in thecontext of ROS-generation in both organelles [5,6,35–38]. Deple-tion of Ca2+-ions in the ER results in ER-stress, which is associatedwith the induction of ROS, autophagy and cell death—processes

that can be counteracted by antioxidants, including NAC [6,36–38].In general, Ca2+-homeostasis in chronic myeloid leukemia cells,like K562 cells, is influenced by the oncoprotein BCR-Abl, reflectedby low ER-Ca2+-levels and a constricted store operated calciumentry (SOCE) [18]. Hence, BCR-Abl could also play a, so far, notcharacterized role in the observed effects which occur followingadrenergic drug treatment in the K562 cell line.

Regarding Ca2+-mobilization we obtained different results forseveral used α1-adrenergic drugs. Surprisingly, experimental eleva-tion of extracellular, respectively intracellular Ca2+ protected the cellsagainst naph-induced toxic effects. This result is in contrast to theobservation that naph induces a short and weak elevation of intra-cellular Ca2+. Regulation of intracellular Ca2+-compartimentalizationis seen as a complex process in which the ER, mitochondria andCa2+-transporters in theplasmalemmaact in concertwitheachotherto maintain the cellular Ca2+-gradient [4,5]. Since we were able toattenuate the toxic action of naph on K562 cells by ionomycinor enhancement of extracellular Ca2+, we hypothesize that naphinterferes with cellular Ca2+-homeostasis by disturbing the balancebetween intracellular Ca2+ elevation and the compensatory influx ofextracellular Ca2+. This hypothesis is in linewith theobservation thatchelating extracellular Ca2+ mimics the actions of naph on growthinhibition and aggregation in K562 cells [1].

Benox induced a maximal elevation of intracellular Ca2+ in K562cells similar as high as the Ca-ATPase inhibitor thapsigargin. A drugcocktail containing benox and thapsigargin did not showa synergisticeffect of the drugs on the maximum amplitude of Ca2+-elevation,suggesting that benox and thapsigargin modulate the same target.As we did not perform assays examining a direct interaction of α1-adrenergic drugs with extracted SERCA—which is the primarytarget of thapsigargin—we could not prove whether benox andother tested α1-adrenergic drugs really bind SERCA-pumps, respec-tively, a protein which is associated with SERCAs, as yet. For thatreasonwe designated the intracellular binding site recognized byα1-adrenergic drugs as thapsigargin sensitive binding site. Aftercombined stimulation of K562 cells with benox and thapsigargin afast raise of intracellular Ca2+ followedbyan immediate slopeof Ca2+

could be observed, reflecting an unknown interaction of thedrugs, which avoids the thapsigargin-induced SOCE [39]. Naph alsodid not influence the height of the benox-evoked Ca2+-peak, butinterfered with the maintenance of the benox-evoked Ca2+-signalin K562 cells. This result strengthens the hypothesis that naphinterferes with the compensatory reaction of K562 cells to maintainCa2+-homeostasis in reaction to intracellular Ca2+-elevation. Theexact molecular mechanism of the interaction of benox and naphremains unclear, so far.

Prazosin was unable to induce a raise of intracellular Ca2+ inK562 cells and also did not interferewith Ca2+mobilization inducedby thapsigargin and naph. However, prazosin also negativelyinfluenced the duration of benox-induced Ca2+-elevation in asimilar manner, which provides an interface between benox,prazosin and naph regarding Ca2+-mobilization. Nevertheless, anenergy driven ion-exchange must be activated by prazosin, sinceprazosin treatment of K562 cells resulted in a sodium azid sensitiveloss of water, indicated by shrinkage of the cells.

In the literature it is well documented that an extensive raise ofintracellular Ca2+, as observed following benox treatment of K562cells, supports apoptosis, mediated by ER-stress and/or Ca2+-overloading of mitochondria [5,35]. Thapsigargin, which inducesCa2+-elevation similar as strong as benox, indeed induces apoptosis

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through Ca2+by induction of ER-stress and activation of caspase 3 inerythroleukemia cells [40].

Modulation of intracellular Ca2+ is also closely linked to erythroiddifferentiation in erythroleukemia cells as well as in physiologicerythroid precursors [7,8].

Prompted by the observation that benox induced a similar(weak) increase of CD41a expression in K562 cells as seen followingprazosin treatment [41], we at first drew the conclusion that benoxmay induce megakaryocytic differentiation without endomitosis.However, in the light of the new data we have now falsifiedthis hypothesis. In fact, it seems that benox, which caused a loss ofGPA+-cells but led to hemoglobinization in concert with hemin,induced erythroid differentiation and subsequently stimulatedapoptosis in these cells. The induction of erythroid differentiationin concert with apoptosis in K562 cells is a common feature of manypro-apoptotic drugs like imatinibmesylate or anthracyclines [42,43].In the case of prazosin the hypothesis of suppression of erythroiddifferentiation could be confirmed, since the percentage of hemo-globinized cells was lowered following prazosin treatment [1].

Overall, our observations strengthen thehypothesis thatCa2+ is acrucial factor for induction of erythroid differentiation in erythro-leukemia cells (Fig. 6) [7,8]. Prazosin, which induces endomitosis inerythroleukemia cells, suppresses hemoglobinization [1] and doesnot evoke Ca2+ -mobilization in K562 cells. On the contrary,hemoglobinization is induced by benox [1]which raises intracellularCa2+. Erythroid differentiation, indicated by the loss of glycophorin-a and hemoglobinization [1], is suppressed by naph which disturbscellular Ca2+-homeostasis. Finally, naph treatment inhibits benox-inducedhemoglobinization [1], reflectedby interference of thedrugswith respect to the maintenance of the Ca2+-signal.

In summary, α-adrenergic drugs are able to modulate theintracellular Ca2+-concentration of erythroleukemia cells througha common, non-adrenoceptor, intracellular binding site. Binding ofadrenergic drugs to this unknown target controls the fate ofhuman erythroleukemia cells towards differentiation, senescenceand cell death.

Acknowledgments

R.F.was supported by a grant of theMedical University of Graz and bythe Franz Lanyar Foundation. Some reagents used in the project wereprovided in cooperation with the NRN grant S93 of the AustrianScience Fund (FWF). Thanks to Dr. Ismene Fertschai for providingFCCP and Maria Teresa Botello Mulet for improving the English text.

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