α1-adrenergic drugs modulate differentiation and cell death of human erythroleukemia cells through...

E X P E R I M E N T A L C E L L R E S E A R C H 3 1 7 ( 2 0 1 1 ) 2 2 3 9 – 2 2 5 1

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Research Article

α1-adrenergic drugs modulate differentiation and celldeath of human erythroleukemia cells through nonadrenergic mechanism

Robert Fuchsa,⁎, Elisabeth Schramla, b, Gerd Leitingerc, d, Ingeborg Stelzer a, e, Nathalie Allarda,Helga Susanne Haasa, Konrad Schauensteina, †, 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 of Molecular Medicine, Medical University of Graz, Harrachgasse 21/7,8010 Graz, AustriadCenter for Medical Research, Core facility Ultrastructure Analysis, Medical University of Graz, Stiftingtalstrasse 24, 8010 Graz, AustriaeClinical 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: [email protected]

† Deceased on May 22, 2007.

0014-4827/$ – see front matter © 2011 Elseviedoi:10.1016/j.yexcr.2011.07.005

A B S T R A C T

Article Chronology:

Received 8 September 2010

Revised version received 1 July 2011Accepted 6 July 2011Available online 14 July 2011

Preliminary data showed that α1-adrenergic antagonists induce apoptosis and a switch towardsmegakaryocytic differentiation in human erythroleukemia cells. To test the hypothesiswhether survival

and differentiation of erythroleukemia cells are under control ofα1-adrenergic signalling,we examinedα1-adrenoceptor expression of erythroleukemia cells and compared the in vitro effects ofα-adrenergicantagonists with those of agonists. We discovered thatα1-adrenergic agonists suppress both erythroiddifferentiation and growth of erythroleukemia cells concomitant with lipofuscin accumulation,autophagy and necrotic cell death. α1-adrenergic agonists also inhibit the in vitro growth ofphysiologic hematopoietic progenitors obtained from umbilical cord blood with high selectivity forthe erythroid lineage. Interestingly, the observed effects could not be related toα1-adrenoceptors, eventhoughagonists andantagonistsdisplayedopposingeffects regarding cellular growthanddifferentiationof erythroleukemia cells. Our data suggest that the effects of α1-adrenergic drugs are related to a non-adrenoceptor binding site, controlling the fate of erythroid progenitor cells towards differentiation andcell death. Since the observed effects are notmediated throughadrenoceptors, the physiologic relevance

of our data remains unclear, so far. Nevertheless, the identification of the still unknown binding site(s)might disclose new insights into regulation of erythroid differentiation and cell death.

© 2011 Elsevier Inc. All rights reserved.

Keywords:

Erythroleukemia cellsErythroid progenitor cellsα1-adrenergic drugs

Cell deathDifferentiation

Introduction

A recent study in our lab showed that α1-adrenergic antagonistsinhibit the growth and induce apoptosis in human erythroleuke-

0.(R. Fuchs).

r Inc. All rights reserved.

mia cells [1]. Furthermore, we observed a suppression of theerythroid phenotype of the cells and a switch towards megakary-ocytic differentiation [1]. The primary objective of this follow upstudy is to investigate, whether the observed effects are dependent

http://dx.doi.org/10.1016/j.yexcr.2011.07.005

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on α1-adrenoceptors, since the pro-apoptotic effect of the α1-adrenergic antagonist prazosin on prostate cancer cells wassuggested to be a non-adrenergic function of the drug [2]. So far,three independent studies report that the growth of the humanerythroleukemia cell line K562 is affected in the presence of α1-adrenergic drugs, namely prazosin [1,3] and phenylephrine [4]although α1-adrenoceptor expression is not formally proven inthis cell line, as yet.

The essential scientific background of our research is theobservation that catecholamines, generally known as potentmodulators of peripheral immune functions [5,6], also influencehematopoiesis and – in particular – erythropoiesis [7–13]. Theimpact of catecholamines on in vitro growth of erythroid cells andin vivo erythropoiesis has been the topic of investigations since theearly 70s of the last century [1,4,7,10,14–16]. For instance,Obayashi et al. elucidated that symphatectiomized rats did notshow altered leukocyte counts in the peripheral blood, butexhibited signs of anemia [14]. Izaguierre et al. demonstratedthat the α1-adrenoceptor antagonist prazosin inhibits erythro-poiesis in mice [15]. Contrary to sympathectomy and adrenergicantagonist treatment, chronic adrenergic stress, for example as aconsequence of trauma, also supports the development of anemia[10,16]. Interestingly, in opposition to erythrocyte counts, leuko-cytes are not changed numerically in the serum of trauma patients[16]. The pronounced susceptibility of the erythroid systemtowards sympathetic dysregulation suggests a modulatory func-tion of the sympathetic nervous system on erythropoiesis[1,4,7,10,14–16]. This hypothesis is in line with the view of anevolutionary interrelation of neuronal and hematopoietic signal-ling mechanisms [5,17,18]. Most studies investigating the impactof the sympathetic nervous system on erythropoiesis focused onβ2-adrenergic signalling pathways, so far. The reason for this isthat erythroid growth is stimulated by β2-adrenergic agonists, butnot, or just under defined conditions, by α-adrenergic agonists[4,7,10]. Although it is known that murine fetal erythroid liver cellsand the cell line human erythroleukemia (HEL) express α2-adrenergic receptors [19,20], the physiologic function(s) of thesereceptors is/are not clarified, as yet.

Parallel to erythropoiesis, a potential adrenergic regulation ofmyelopoiesis is suggested by Maestroni and co-workers [8,9].These authors discovered that myelopoiesis is under an inhibitoryadrenergic control, mediated through α1b-adrenoceptors [8,9].This result could be strengthened previously in our lab through theobservation that the α1-adrenergic agonist oxymetazoline, butalso the β2-adrenergic agonist terbutaline inhibit the in vitrogrowth of myeloic colony forming units isolated frommurine bonemarrow [13].

In the light of previous studies, our findings regarding theeffects of α1-adrenergic antagonists on erythroleukemia cellsprompted us to suggest that, so far, neglected α1-adrenoceptorscould possess fundamental impact on both survival and differen-tiation of immature erythroid progenitor cells. To test thishypothesis, we used the erythroleukemia cell lines K562 andHEL again [1,21–23], as well as physiologic hematopoietic stemand progenitor cells obtained from human umbilical cord blood. Inorder to induce a state of chronic adrenergic stress on leukemiacells respectively hematopoietic progenitors, cell cultures weretreated with high doses of α-adrenergic agonists. Furthermore,erythroleukemia cell cultures were treated with both agonists andantagonists in order to observe, whether agonist actions can be

blocked by antagonists or, vice versa, antagonist actions arereversible by agonist treatment. In addition, expression of all threeknown α1-adrenergic receptor subclasses (ADRA1A, ADRA1B andADRA1D) was detected in the cell lines K562 and HEL usingTaqMan® RT-PCR assays.

In the present study we show that α1-adrenergic agonistsinhibit the in vitro growth of erythroid progenitor cells and suppressboth differentiation as well as growth of erythroleukemia cells,concomitant with the induction of necrotic cell death. Interestingly,the observed effects are not related to known α1-adrenoceptors,although α1-adrenergic agonists and antagonists exerted opposingeffects regarding growth and differentiation of erythroleukemiacells. Thus, the physiologic respectively pathophysiologic signifi-cance of the observed effects in the context of a relationshipbetween sympathetic signalling and erythropoiesis remains unclear,so far. Nevertheless, our study shows that an unknown target,which exhibits affinity to several α1-adrenergic drugs, holds anessential position in the maintenance of cellular homeostasis inerythroid cells.

Materials and methods

Adrenergic drugs

To simulate the effects of catecholamines, the adrenergic agonistsnaphazoline HCl (naph, α1 [24]), oxymetazoline HCl (oxy,α1/partial α2 [24,25]) or clonidine HCl (α2 [24]) – each dissolvedin cell culture medium –were added to cultures of erythroleukemiacells or to cord blood-colony-forming-unit (CFU)-assays. Theadrenergic antagonists benoxathian HCl (benox, α1), prazosinHCl (α1) or yohimbine HCl (α2) were added to cell cultures asaqueous solutions; alone or in combination with agonists. Alladrenergic drugs were obtained from Sigma (St. Louis, MO/USA),except oxy which was purchased from ICN Biomedicals (Aurora,OH/USA).

CFU-assays with umbilical cord blood (UCB) derivedmononuclear cells (MNCs) and cultivation of leukemia cells

Umbilical cord blood was obtained from the Institute of Obstetrics atthe University Clinic of Graz with consent of the mothers. Blood wascollected under sterile conditions at night before experimentalprocedure, not extending 14 h before cell preparation. As anticoag-ulant ammonium heparin was used. Mononuclear cells were isolatedby means of density centrifugation using Histopaque 1640 (Sigma)according to the instructions of the producer. Subsequent to densitycentrifugation cells were washed twice with calcium–magnesiumfree phosphate buffer (CMF-PBS) and re-suspended in MEM-alpha(Invitrogen/Gibco, Paisley, UK) supplemented with 15% fetal calfserum, glutamine and streptomycin/penicillin. For cultivation of stemand progenitor cells, the methylcellulose based medium MethocultGFH4434 (StemCell Technologies Inc., Vancouver, Canada)was used.1×E4 MNCs per ml Methocult were plated in duplicates in 35 mmdishes in an incubator at 37 °C, 5% CO2 in a fully humidifiedatmosphere. Following 13–14 days of incubation, the dishes werescreened for colony forming unit — granulocyte-monocyte (CFU-GM)-colonies, burst forming unit erythrocyte (BFU-E)-colonies andCFU-erythrocyte (CFU-E)-colonies. BFU-E,- and CFU-E-colonies werecounted together as erythroid colonies. Subsequent to colony

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counting, the semi liquid medium was re-suspended in phosphatebuffer to obtain ahomogenous cell suspension. The total cell counts ofthese suspensions were assessed with a CASY® Cell Counter &Analyzer (Innovatis, Reutlingen, Germany).

Human erythroleukemia (HEL) cells (DSMZ, Braunschweig,Germany), K562 cells (ATCC, Manassas, VA/USA) and Thp-1 cells(ATCC) were maintained in RPMI 1640 medium (Lonza, Verviers,Belgium) supplemented with glutamine, penicillin/streptomycinand 10% fetal calf serum. Cell cultures were kept in an incubatorat 37 °C, 5% CO2 in a fully humidified atmosphere. For testingproliferation, both HEL and K562 cells (start cell numbers 1×E4 or2×E4 cells/ml) were incubated in 24-well or 12-well plates two orthree days with adrenergic drugs in triplicates. Proliferation andviability of the cells were assessed with the CASY® Cell Counter &Analyzer.

Analysis of α1-adrenergic receptor-expression usingTaqMan® RT-PCR

To assess the expression ofα1-adrenoceptors ADRA1A, ADRA1B andADRA1D in human erythroleukemia cells, TaqMan® RT-PCR assays,designed by Applied Biosystems (AB, Foster City, CA/USA), wereused. To quantify adrenoceptor expression, the expression of thereference gene ACTB was analyzed parallel to adrenoceptors. Aspositive controls for the expression of α1-adrenoceptors, thetranscriptome of the human leukemia cell line Thp-1 and humanpost mortem heart tissue with normal diagnosis were analyzed[26–29]. Human heart tissue (samples from anterior and posteriorwall of the myocardium) was obtained from the Biobank Graz withconsent of the local ethical committee (decision number: 23–410ex 10/11). Total RNA from the cell lines K562, HEL, Thp-1 and hearttissue was extracted using TRI-Reagent RT (MRC Inc., Cincinnati,OH/USA) in accordance to the manufacturer's protocol. ExtractedRNA was quantified and analyzed for the 260/280 ratio by aNanoDrop ND-1000 spectrophotometer (Peqlab, Erlangen, Germa-ny). Quality of extracted RNA was further examined by RNA-gelelectrophoresis. cDNAs were generated, using the High CapacitycDNA Reverse Transcription Kit (AB). RT-PCR according to theTaqMan® RT-PCR assay-protocol was performed on an iCycler iQ™or a CFX96™ — Real-Time PCR Detection System (Bio-RadLaboratories, Hercules, CA/USA). Each sample was measured atminimum in duplicates. To improve the sensitivity of the RT-PCRassay, an optional pre-amplification step was performed prior toRT-PCR, because ADRA1-transcripts show low abundance in manytissue types [30]. Pre-amplification was done related to theTaqMan® PreAmp Master Mix Kit Protocol provided by AB. Forpre-amplification, the TaqMan®-assays of α1-adrenoceptors andbeta-actin were pooled and diluted with TE-buffer to reach a final1:100 dilution. The 50 μl pre-amplification reactions contained12.5 μl of the diluted assays, 2 mM MgCl2 [31], 100 ng of cDNA,25 μl PCR-Master Mix S (Peqlab) and were filled up to the finalvolume with PCR grade water before running ten amplificationcycles in a standard thermo cycler. In the following RT-PCR, 5 μl ofthe 1:5 diluted pre-amplification products were used in each(20 μl) reaction.

Transmission electron microscopy

For ultrastructure analysis, K562 cells were cultivated with orwithout 200 μM naph for 48 h, washed once in phosphate buffer

and were prepared for electron microscopic inspection using astandard protocol as described previously [1]. Cells were examinedwith a Zeiss EM 902 transmission electron microscope.

Measurement of caspase 3-activity

In order to detect the induction of apoptosis in erythroleukemiacells by adrenergic drugs, the activation of caspase 3 was analyzed,using the FITC-Active Caspase 3 Apoptosis Kit of Becton Dickinson(BD, San Diego, CA/USA) as described previously [1]. The assaywasperformed according to the protocol of the producer, followingincubation of cells with/without adrenergic drugs at standard cellculture conditions. Subsequent to cell preparation and antibody-staining for active caspase 3, cells were analyzed with a BD-FACScan flow cytometer using CELLQuest software (BD).

Detection of autophagosomes and lysosomes

In order to detect the induction of autophagy in erythroleukemiacells, indirect immune fluorescence assays against the autophago-some related protein LC3B (Atg8) [32] were performed. After K562cells were treated with adrenergic drugs for 48 h, cells wereharvested, washed once with phosphate buffer and were spinnedon slides and fixated with methanol (−20 °C) for 5 min. Afterdrying, slides were stored at−20 °C until performing the antibodystaining procedure. Cells were incubated in a moist chamber witha primary rabbit anti LC3B antibody (Cell Signalling Technology,Danvers, MA/USA), diluted in phosphate buffer supplementedwith 0.3% Igepal CA-630 (Sigma) and 5% normal goat serum(Jackson ImmunoResearch, West Grove, PA/USA) over night at4 °C. After washing three times with phosphate buffer, cells werestained with a secondary FITC-labelled goat anti rabbit antibody(Sigma), diluted in phosphate buffer containing 0.3% Igepal CA-630 for 90 min in the dark at room temperature and washed againtwice. Following counterstaining with 4′,6-diamidino-2-phenylin-dol (DAPI, Sigma) and one further washing step, slides werecovered with mounting medium (INOVA-Diagnostics, San Diego,CA/USA) and cover slips. Afterwards cells were inspected with aLeica-DM 4000 fluorescence microscope for autophagosomes.

For analysis of lysosomes respectively intracellular pH, cellswere stained with acridine orange (AO, Sigma), as described byPacheco et al. [33]. Acridine orange (AO) diluted in aqua bidest(1 mg/ml) was added directly into K562-cells containing cellculture vessels to reach a final AO concentration of 5 μg/ml. Cellswere stained for 15 min at 37 °C, washed twice with RPMI-1640and were directly examined with a Leica-DM 4000 fluorescencemicroscope. The (green) nuclear AO stain was viewed using a filterwith the excitation bandpass of 480/40 nm (Leica Filter Cube L5)and the orange AO stain of acidic organelles (mainly lysosomes)was viewed using a filter with the excitation bandpass of 515–560 nm (Leica Filter Cube N2.1).

Detection of polyploidy in K562 cells

Polyploidy of K562 cells was assessed by microscopy andpropidium iodide (PI) staining. Cells were treated 72 h withnaph (100 μM), prazosin (10 μM) or combinations of both drugs.For microscopic analysis cells were harvested, spinned on slidesand stained with May-Grünwald-Giemsa using a standard proto-col. In a blindedmanner 300 cells per condition were analyzed and


the percental distribution of cells with polylobulated nuclei wasdetermined. For PI staining, cells were harvested, washed once inice cold CMF-PBS and fixated in 70% ice cold ethanol for 1 h. Untilstaining with PI, cells were stored at −20 °C in the fixative. Beforesuspending in the PI staining solution on the basis of CMF-PBScontaining 50 μg/ml PI (Sigma), 0.1 mg/ml RNAse A (Sigma), 0.1%sodium-citrate and 0.1% Igepal CA-630 (Sigma), cells were washedagain. Cells were incubated protected from light in a water bath for15 min at 37 °C and analyzed immediately with a FACSCalibur flowcytometer (BD, San Jose, CA/USA).

Analysis of glycophorin a and hemoglobin

Erythroid differentiation of erythroleukemia cells was assessed byanalyzing the expression of the erythroid marker glycophorin a(GPA, CD235a) using a PECy5 labelled mouse anti-human GPAantibody (BD-Pharmingen) by flow cytometry and by detection ofhemoglobinization by means of cytochemistry. Cells, obtainedfrom day 14 CFU-assay dishes, were analyzed for the expression ofCD45 and CD34 using the antibody-combination CD45-FITC/CD34-PE of BD (San Jose, CA/USA), parallel to GPA. Flow cytometryanalyses were performed on a BD-FACScan using CELLQuestsoftware (BD).

To stimulate hemoglobin synthesis in K562 cells, cells wereincubated with 20 μM or 40 μM hemin (Sigma) for 72 h [21–23];alone or in presence of adrenergic drugs. Hemoglobin synthesiswas monitored by benzidine staining. Subsequent to cultivation,cell suspensionswerewashed twice and approximately 2×E5 cellswere re-suspended in 10 μl phosphate buffer and mixed with thesame volume of benzidine staining solution (2 mg/ml benzidinedihydrochloride (Sigma) in 0.5% acetic acid+5 μl 30% hydrogenperoxide/ml). The benzidine staining solution was always pre-pared freshly for each experiment. After cells were incubated for10 min at room temperature, cells of each condition wereinspected microscopically in a hemacytometer. The cells in thehemacytometer were photographed at 100× magnification andwere classified for being benzidine positive or negative (seeFig. 5D) using the cell counter of the free software Image J(National Institutes of Health). About 1000 cells/condition wereanalyzed in all of the experiments.

Statistics

Results are expressed as mean values plus or minus standarddeviation. Data were analyzed by the unpaired Student's t test orby One Way ANOVA using Sigma Plot 11.0. p-values equal or lessthan 0.05 were considered as significant.

Results

Expression of known α1-adrenoceptors is absent in humanerythroleukemia cell lines K562 and HEL

Since it is not formally proven whether the cell lines K562 and HELexpress α1-adrenoceptors, RT-PCR expression analysis was per-formed in order to screen for α1-adrenoceptor transcripts.Surprisingly, the used TaqMan®-gene expression assays for α1-adrenoceptors (ADRA1A, ADRA1B and ADRA1D) were negative forall three subclasses in the erythroleukemia cell lines HEL and K562

(Fig. 1A). Furthermore, we did not detect ADRA1-signals followinga 30 h treatment with adrenergic agonists or antagonists in K562cells (not shown). Even with pre-amplification, we obtained nopositive signals in all tested conditions. As anticipated, we wereable to prove the expression of ADRA1A, ADRA1B as well asADRA1D in tissue probes from the anterior and posterior wall ofthe human heart with our assays (Fig. 1A). In the Thp-1 cell line wedetected just a weak positive signal for ADRA1B, which wassuccessfully enhanced using the described pre-amplificationprotocol (Figs. 1B/C).

α1-adrenergic agonists inhibit the growth and inducenecrotic cell death in human erythroleukemia cells

As we have observed that α1-adrenergic antagonists induceapoptosis and differentiation in erythroleukemia cells in theabsence of α1-adrenoceptors [1], we were interested to investi-gate, whether erythroleukemia cells respond to α-adrenergicagonist treatment, as well. K562 and HEL cells were incubatedeither three days (K562) or five days (HEL) with increasingconcentrations of the adrenergic agonists naphazoline (naph),oxymetazoline (oxy) or clonidine. Subsequent to cultivation, weanalyzed cell numbers and viability of the cells by means of aCASY® cell counter. The α1-adrenergic agonists naph and oxyinhibit the growth of both cell lines and induce cell death at highdrug concentrations (200 μM), whereas clonidine does notinfluence the growth of both tested cell lines (Fig. 2A). Interest-ingly K562-, and HEL cells exhibit different sensitivity towardstreatment with naph (Figs. 2A/B). Whereas naph treatment of HELcells for three days has no influence on growth and viability of thecells (not shown), the proliferation and viability of K562 cells isdrastically diminished at a concentration of 200 μM (Figs. 2A/B).Following five days of cultivation with naph, the proliferation ofHEL cells is negatively affected too, but not as strong as observed inthe K562 cell line (Fig. 2A).

Parallel to growth inhibition, K562 cells, but not HEL cells,change their growth behavior in the presence of 200 μM naph.Normally growing in suspension or slightly attached to the surfaceof the culture vessel, 200 μM naph or oxy treated K562 cells formcell aggregates, detectable following a 48 h period of cultivation(Fig. 2C). As Ca2+ is a known co-factor for several adhesionmolecules, we tested whether naph-induced aggregation dependson Ca2+-ions. For this purpose, the selective calcium chelatorBAPTA was added to untreated and naph treated K562 cultures.Interestingly, chelation of Ca2+-ions alone mimics the growthinhibitory and aggregation effect of naph and further enhancesnaph-induced aggregation (not shown).

In addition to aggregation of K562 cells after treatment with200 μM naph, we also found that many treated cells looked likespheres, filled with vacuole-like structures (Fig. 2C). The hypothesisof an underlying autolytic process in these cells could be confirmedby transmission electron microscopy (TEM) and by a fluorescencebased assay for detection of autophagy (Figs. 2D/E).

By means of TEM we observed several stages of autophagy,characterized by the appearance of autophagosomes and hugetelolysosomes (lipofuscin vesicles) in naph (200 μM) treated cells(Fig. 2D). We could not detect prevalent signs of apoptosis.Furthermore, some naph treated cells exhibit granular structures(Fig. 2D IV) attached to particular sections outside of the plasmamembrane, reminiscent to previously described exosomes in the

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Fig. 1 – Human erythroleukemia cell lines K562 and HEL do not express α1-adrenoceptors. Expression of transcripts coding forα1-adrenoceptors ADRA1A, ADRA1B and ADRA1D as well as beta-actin (ACTB) was evaluated using Taqman® -RT-PCR assays in thecell lines HEL, K562 and Thp-1 as well as in tissue of the anterior wall of human heart. A: K562 and HEL cells are negative fortranscripts of α1-adrenoceptors. As expected, we could detect transcripts for all known α1-adrenoceptors types in human hearttissue. B: RT-PCR-amplification curves obtained in Thp-1 cells testing for the expression of ADRA1A, ADRA1B, ADRA1D and ACTBwith prior pre-amplification of respective cDNAs. A representative result of a total of three experiments is shown. C: CP values ofevaluated genes, respectively the ΔCP value of ADRA1B related to ACTB, obtained in Thp-1 cells are shown in the table. n=3.


K562 cell line [34]. Naph treated cells also exhibit an accumulationof mitochondrial granules, which suggests the participation ofmitochondria in the naph-induced effects in K562 cells (Fig. 2DIII/IV). Immunofluorescence assays against the autophagy markerLC3B [32] confirmed the augmented formation of autophagosomesin K562 cells following naph treatment (Fig. 2E). In naph treatedcells autophagosomes are enlarged in comparison to untreatedcontrols. Furthermore, naph treated K562 cells show a sporadiccircular enrichment of LC3B signals at the plasmalemma (Fig. 2E).Further experiments revealed that the induction of autophagy inK562 cells is not restricted to adrenergic agonists, but is also inducedby the adrenergic antagonists prazosin and benoxathian (benox;Supplementary Information, Fig. S1). Especially in prazosin-treatedcells we detected huge autophagophore-like LC3B+ structures.

In order to confirm the non apoptotic mechanism of naph-induced cell death as indicated by TEM analysis, the activationstate of caspase 3 was analyzed following 24 h, 48 h and 72 hincubation of K562- and HEL cells with naph. We found that, incontrast to adrenergic antagonists, the adrenergic agonist naphinduces just a slight activation of caspase 3 in both tested cell linesin comparison to untreated controls (K562: Fig. 2F, HEL: notshown). Furthermore, no enhanced nuclear fragmentation isobserved following naph treatment in both tested cell lines, asshown by separate DAPI staining and fluorescence assays (Fig. 2E).

In the analysis of lysosomes we discovered that acridine orangeinduces a diffuse orange colour of naph treated K562 cells underthe fluorescence microscope, indicating acidification. In untreatedcells we observed small orange individual structures, representinglysosomes and other acidic organelles (Fig. 3) [33].

Naphazoline counteracts prazosin-induced growth inhibitionand caspase 3 activation in K562 cells

In order to assess interferences betweenα1-adrenoceptor antagonist-and agonist actions in erythroleukemia cells, the antagonists benox,prazosin or yohimbinewere added toK562 cell cultures togetherwithnaph. Growth and viability of the cells were analyzed followingcultivation. The result of this screening was that neither differentconcentrations of benox, nor prazosin or yohimbine can attenuate thetoxic effect of 200 μMnaph on the growth of K562 cells (not shown),confirming the non α1-adrenoceptor mediated effects of the drugs.But surprisingly, less toxic naph concentrations (50 μM, 100 μM) canattenuate prazosin-induced growth inhibition (Fig. 4A) and theprazosin-induced increase of cell size (Fig. 4C) inK562 cells. Activationof caspase 3 was further analyzed following cultivation of K562 cellswith either prazosin or combinations of prazosin andnaph, in order toassess whether naph suppresses prazosin-induced apoptosis in K562cells. The result of this analysis was that naph is able to suppressprazosin-induced caspase 3 activation in K562 cells (Fig. 4B).

Naphazoline inhibits both megakaryocytic and erythroiddifferentiation of human erythroleukemia cells

To examine whether the effect of prazosin to induce signs ofmegakaryocytic differentiation can be influenced by naph, K562cells were treated together with prazosin (10 μM) and naph(100 μM). Subsequent to cultivation, cells were screened for theappearance of cells with polylobulated nuclei. We could prove thatnaph, which provides protection against prazosin-induced growth


inhibition and apoptosis, also significantly reduces the number ofcells with polylobulated nuclei (Figs. 4C–E). Staining withpropidium iodide confirmed this observation (Figs. 4C–E). 72 hprazosin treatment of K562 cells results in polyploidization up to16 N, which is efficiently suppressed by parallel naph treatment.

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To assess erythroid differentiation of erythroleukemia cells, theexpression of the erythroid marker GPA and hemin-inducedhemoglobinization were analyzed following treatment withnaph. In the K562 cell line, but not in the naph resistant HEL cellline, the expression of GPA is decreasing dose-dependently

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Fig. 3 –Naphazoline induces acidification in K562 cells. Acridine orange (AO) staining was performed in order to assess acidificationrespectively lysosomal integrity. K562 cells were treated 48 h with/without addition of 200 μM naphazoline. The fluorescencefigures are overlays, generated from figures of the nuclear staining of AO and the staining of acidic organelles. Naphazolinetreatment of K562 cells induces a diffuse orange staining of K562 cells, indicating acidification.


following naph treatment (Figs. 5A/B) similar as observed withα1-adrenergic antagonists [1]. Converting the relative numbers ofGPA− respectively GPA+ cells, as determined by FACS analysis, toabsolute cell numbers per well, an enrichment of GPA− cells can beseen with a maximum at 100 μM naph, along with a dose-dependent loss of GPA+ cells (Fig. 5B). This accumulation of GPA−

cells could not be observed following antagonist treatment.In order to stimulate hemoglobinization, K562 cells were

treated with hemin, which we added to cell cultures alone or incombination with adrenergic drugs. In accordance to data in theliterature [21–23], hemin induces the appearance of benzidinepositive K562 cells during 72 h cultivation (image in Fig. 5D).20 μM hemin does not significantly interfere with the growth

Fig. 2 – α1-adrenergic agonists inhibit the growth, induce cell deathcells. A: Human erythroleukemia cells were cultivated three (K562)(naph), oxymetazoline or clonidine. Data are presented as the relat(=100%). All data are shown as means of at minimum three experimB: The cell lines K562 and HEL show different sensitivity against naC: Naph treatment (200 μM) induces the formation of cell aggregatpicturewithin the naph-figure displays an exemplary cell from a napsmall proportion of cells, looking like spheres, filled with vacuole-(200 μM) treated K562 cells. I: Untreated control, II-IV: Naph treatevacuolization, huge telolysosomes (white arrow in II), mitochondrappear in K562 cells. IV: Naph treatment induces signs of autophagautophagophores (black arrows). Some cells show a bulk of small vdescribed as exosomes (Ex).M:mitochondria, N: nucleus, T: telolysoNaph treatment (200 μM) induces the formation of LC3B+ autophagat a closer look in II. II: 48 h naph treatment (200 μM) induces the acK562 cells (arrow). F: Analysis of active caspase 3 in naph treated Kmarginal activation of caspase 3 in K562 cells.

behavior of K562 cells, but induces a slight increase of benzidinepositive cells (Supplementary Information, Figs. S2A/B). 40 μMhemin inhibits the growth (Fig. 5C) and lowers the viability of thecells, but induces a higher percentage of benzidinepositive cells than20 μM hemin (Supplementary Information, Fig. S2B). The combina-tion of hemin and naph does not result in a synergistic effect oncellular proliferation of K562 cells (Fig. 5C). Naph treatment dose-dependently and significantly (100 μM: p<0.05, 200 μM: p<0.01)inhibits the generation of hemoglobinized (benzidine positive) cellsin combination with 40 μM hemin (Fig. 5D).

α1-adrenergic antagonists also influence the percentage ofbenzidine positive cells following hemin treatment, but in a differentmanner than naph (Supplementary Information, Fig. S2). When

and influence the growth behavior of human erythroleukemiaor five (HEL) days with the α-adrenergic agonists naphazolineive number of viable cells in comparison to untreated controlsents per each condition (except clonidine in K562 cells, n=2).ph treatment, concerning viability. HEL: n=4, K562: n=3.es in K562 cultures (original magnification 100×). The smallh treated culture (originalmagnification: 400×), representing alike structures. D: Electron microscopic analysis of 48 h naphd K562 cells. II/III: Following naph treatment cytoplasmicial granules (black arrows in III) and autophagosomes (III)ocytosis in K562 cells as indicated by the appearance ofesicles at the cytoplasmic membrane (white arrows), previouslysomes. E: Immunofluorescence staining of LC3B in K562 cells. I:osomes in K562 cells. Themarked areas in the figures are showncumulation of LC3B+— signals at the cytoplasmic membrane of562 cells. In contrast to benoxathian, naph induces just a

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Fig. 4 – Naphazoline attenuates the pro-apoptotic and pro-endomitotic effects of prazosin in the K562 cell line. A: K562 cells werecultivated 72 h with naphazoline (naph) or prazosin (prazo) alone or with combinations of both drugs. Subsequent to cultivation,cell numbers were assessed with a cell counter. Data are represented as the relative number of living cells in comparison tountreated controls (=100%). n=5, *: p<0.05 according to unpaired t-test. B: Activation of caspase 3 in K562 cells after 48 hcultivation with prazo with or without combined addition of 100 μM naph. n=3, *: p<0.05, ***: p<0.001 according to unpairedt-test. C: May-Grünwald-Giemsa stained K562 cells following treatment with prazo, naph or a combination of prazo and naph incomparison to untreated control. Original magnification 200×. As described previously, prazo treatment induces the appearance ofhuge, polylobulated nuclei in K562 cells (arrows), identified as cells exhibiting signs of megakaryocytic differentiation. Napheffectively abrogates the prazo-stimulated formation of cells with polylobulated nuclei. The histograms in the left corner of therespective picture indicate the DNA content of the respective cells by means of propidium iodide (PI) staining. D: Statistics of theappearance of cells with polylobulated nuclei following naph treatment. May-Grünwald–Giemsa stained cells were screened forpolylobulated nuclei. n=5, **: p<0.01 according to unpaired t-test. The image in the graph displays a couple of huge cells withpolylobulated nuclei mixedwith small cells with unaltered nuclei at a originalmagnification of 630×. E: Statistics of DNA-content ofnaph and prazo treated K562 cells. Control, Naph: n=3, Prazo, Naph+Prazo: n=4, *: p<0.001 according to One Way ANOVA.


prazosin is added together with hemin (40 μM) to cell cultures, thegrowth behavior of these cells is not altered in comparison to cellstreatedwith prazosin alone, whereas hemoglobinization of the cells is

significantly (p<0.05) inhibited (Supplementary Information, Fig. S2).We still observed the appearance of polylobulated nuclei in K562 cells,indicating no interference of hemin with the process of endomitosis.

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In contrast to prazosin, benox shows a synergistic effect withhemin on K562 cells, inhibiting growth and inducing differentia-tion. With 40 μM hemin, the cytotoxity of benox is enhanced in asynergistic manner (Supplementary Information, Fig. S2). Surviv-ing cells show a significantly (p<0.05) enhanced percentage ofhemoglobinization. With 20 μM hemin, benox (10 μM) enhancesthe capacity of K562 cells to produce hemoglobin by a factor ofabout four (Supplementary Information, Fig. S2). To assess thereversibility of the benox-induced hemoglobinization by naph(50 μM or 100 μM), both drugs (benox: 10 μM) were combinedand added to hemin (20 μM) stimulated K562 cells. Indeed, naphdose-dependently and significantly (p<0.05) abrogates thehemoglobinization induced by benox/hemin (SupplementaryInformation, Fig. S2C). In comparison to cells treated with benox/hemin only, growth inhibition is enhanced by naph, without

further decrease in viability of the cells (Supplementary Informa-tion, table in Fig. S2C).

α1-adrenergic agonists inhibit the growth of hematopoieticprogenitor cells obtained from human cord blood with highselectivity for erythroid progenitors

Hematopoietic stem and progenitor cells derived from humanumbilical cord blood were used as a second model system toanalyze the effects of adrenergic agonists on in vitro hematopoi-esis. Following two weeks of incubation, CFU-assay-dishes werescreened for colony growth. Already by macroscopic viewing ofthe dishes, we observed significant differences. In naph or oxytreated dishes we detected fewer red spots, formed by colonies ofthe erythroid lineage (Fig. 6A). This first result could be confirmed

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Fig. 6 – α1-adrenergic agonists selectively inhibit the in vitro growth of UCB-derived erythroid progenitor cells. A: UCB-derivedprogenitor cells were cultivated 14 days in medium containing the adrenergic agonists naphazoline (naph), oxymetazoline (oxy) orclonidine (clo). Subsequent to cultivation, colony growth was analyzed. Naph treated dishes exhibit less erythroid colonies (BFU-E)in comparison to untreated controls (upper row). Existing BFU-E are less distinctive in size and morphology (lower row).B: Statistics of colonies per CFU-dish. Relative numbers of BFU-E and myeloic colonies (CFU-GM) are shown in comparison tountreated controls (C=100%). C: n=10, naph [10 μM]: n=5, naph [100 μM]: n=9, oxy [10 μM]: n=4, oxy [100 μM]: n=4, clo[10 μM]: n=3, clo [100 μM]: n=4, *: p<0.001 according to One Way ANOVA. C: Total cell counts following 14 day cultivation ofUCB-derived progenitor cells. C (=100%): n=13, naph [10 μM]: n=8, naph [100 μM]: n=13, oxy [10 μM]: n=5, oxy [100 μM]:n=5, clo [10 μM]: n=4, clo [100 μM]: n=6, **: p<0.01, ***: p<0.001 in comparison to untreated controls according to One WayANOVA. D: Flow-cytometry analysis of naph-treated UCB-derived progenitor cells. Cells of day 14 CFU-assays were harvested andanalyzed for the expression of CD45, GPA and CD34. Naph treated cells exhibit an altered light scatter characteristics and anenrichment of CD45 positive cells, concomitant with a decline of erythroid (GPA+) cells. The black curves in the histograms CD45,GPA and CD34 represent the respective fluorescence signal of unstained cells. E: Statistics of the leukocyte (CD45+) fraction of day 14UCB-cultures. F: Statistics of the erythroid (GPA+) fraction of day 14 UCB-cultures. E/F: C: n=6, naph [10 μM]: n=6, naph [100 μM]:n=6, oxy [10 μM]: n=4, oxy [100 μM]: n=4, clo [10 μM]: n=4, clo [100 μM]: n=4, *: p<0.001 according to One Way ANOVA.



by analyzing the dishes under the microscope. In dishes with naphor oxy containing medium, the formation of erythroid colonies isinhibited, whereas the generation of CFU-GM derived coloniesseems to be less affected (Fig. 6B). The existing red colonies in theα1-adrenergic agonist treated dishes are smaller in size and showless contrast and color (Fig. 6A). The inhibitory effect of oxy(100 μM) is significantly (p<0.001) more pronounced than theinhibitory effect of naph at the level of colony number (Fig. 6B).Oxy (100 μM) almost extinguishes erythroid colony growth,whereas naph shows a milder colony-reducing effect. Comparedtoα1-adrenergic agonists, theα2-adrenergic agonist clonidine hasno, or just a minimal effect on colony growth (Fig. 6B).

Analysis of cell counts revealed that the total cell number perdish is significantly decreased by either treatment with naph oroxy, whereas the effect of clonidine is mild (Fig. 6C).

Flow cytometry analysis of cells isolated from day 14 CFU-dishes exhibited a large heterogeneity of cellular composition dueto different types of lineages (erythroid versus myeloic lineage)and also several stages of differentiation.

Analysis of re-suspended colonies further confirmed theimpression of the selective inhibition of erythroid growth, sincethe CD45+ cells fraction is significantly (p<0.001) enriched incomparison to the GPA+ fraction in naph or oxy (respectively100 μM) treated dishes (Figs. 6D–F). In comparison to untreatedcontrols, cells harvested from naph or oxy treated dishes also showhigher granularity (side scatter/SSC), and enhanced cell size(forward scatter/FSC) (Fig. 6D). The mean GPA expression oferythroid cells per se and the overall mean expression of the CD34stem cell marker are not altered by adrenergic agonist treatment(Fig. 6D).

Discussion

The studies of Myklebust at al. [35] and Van Lindern et al. [36]suggest a so far unknown G protein-coupled receptor (GPCR) witha Gαq subunit, controlling erythroid differentiation in concertwith the EPO-receptor. In the light of previous data, showingthat both differentiation and viability of human erythroleukemiacells are affected by α1-adrenergic antagonist treatment [1,3] anddue to the fact that α1-adrenergic receptors belong to GPCR witha Gαq subunit [37], we were optimistic that we had found thisneedle in the haystack.We thus aimed to detect ADRA1-expressionin erythroleukemia cells using TaqMan®-RT-PCR because of highspecificity and sensitivity and for the simple reason that nospecific commercial antibodies for human ADRA1 are available[38]. However, human erythroleukemia cells were negative fortranscripts of all three known subtypes of ADRA1. Therefore, α1-adrenergic receptors are, at least in erythroleukemia cells, nosuitable candidates for the role as signal amplifier of the EPO-receptor. The dependence of the observed effects on α2-adrenergic receptors can also be ruled out, since the highly α2-selective adrenergic drugs yohimbine [1], respectively clonidinehad just weak effects in comparison to α1-adrenergic drugs. Theα1-adrenergic drugs must therefore bind to a hitherto unknownnon adrenoceptor-binding site. This is in line with the observa-tion that α1-adrenergic antagonists – including prazosin –

induce apoptosis in different types of human cells through anon adrenoceptor mediated mechanism [2,39–41]. Our observa-tions suggest that the target of α1-adrenergic antagonists is also

a key factor for both survival and differentiation of leukemic aswell as physiologic erythroid precursor cells. So far, theunderlying mechanisms of the apoptosis inducing properties ofα1-adrenergic antagonists remain mainly unknown. Some dataindicate that ER-stress might be involved in the pro-apoptoticeffects of adrenergic antagonists [39], whereas other studiessuggest a role of NF-kappaB and the signalling system of TGF-β[40,41]. In our current study we could demonstrate for the firsttime that the α1-adrenergic agonist naph is able to suppress theadrenoceptor independent pro-apoptotic effect of prazosin. Naphper se induced necrotic cell death at high concentrations andinterfered with the induction of differentiation in erythroleuke-mia cells. Because of the contrary effects of agonists andantagonists regarding cell death and differentiation we hypoth-esize a common target of the drugs. Interestingly, antagonists aswell as the agonist naph induced the formation of autophago-somes in erythroleukemia cells, which is a clear indication for acellular stress response [42].

Since we solely used synthetic adrenergic drugs in our studyand we found no correlation between the observed effects of α1-adrenergic drugs and adrenoceptors, the physiologic significanceof our results remains unclear, so far. Nevertheless, our in vitroresults show interesting parallels with results obtained in vivo inother studies. On the one hand, the group around Livingston et al.discovered that the growth of rat erythroid colonies is inhibitedfollowing supraphysiologic doses of exogenous norepinephrine inthe animals [43]. On the other hand, Izaguirre et al. revealed aninhibitory effect of prazosin on erythropoiesis in normoxic orhypoxic mice [15].

From the pathophysiologic point of view indicating thatadrenergic stress induces anemia [16], our results suggest thatthis effect is not mediated throughα1-adrenoceptors on erythroidprogenitor cells. Since high concentrations of clonidine justslightly influenced growth of UCB-derived progenitor cells anderythroleukemia cells, also the role of α2-adrenoceptors remainsquestionable in this context.

Our next objective is to visualize the unknown targetrecognized by α1-adrenergic drugs in erythroid cells and to testwhether this target interacts with norepinephrine, as well.Furthermore, we aim to examine, whether the effects of α1-adrenergic drugs on growth and differentiation of humanerythroleukemia cells are related to calcium signaling. Cellularcalcium is closely related to the signaling of erythropoietin,erythroid differentiation, cell death and survival of physiologicand leukemic erythroid progenitor cells [44–48].

A strategy for visualization and possible further identificationof the non-adrenoceptor-targets of α1-adrenergic drugs by meansof the fluorescent α1-adrenergic antagonist BODIPY® FL-Prazosin[49] in erythroleukemia cells is presented in the following paper[50]. Furthermore, evidence is provided that Ca2+ is a contributingfactor in the effects of α1-adrenergic drugs on erythroleukemiacells [50].

Supplementary materials related to this article can be foundonline at doi:10.1016/j.yexcr.2011.07.005.

Acknowledgments

R.F. was supported by a grant of theMedical University of Graz andby the Franz Lanyar foundation. Some reagents used in the project



were provided in cooperation with the NRN grant S93 of theAustrian Science Fund (FWF).

Thanks to Elke Schwarzenberger, Brigitte Poncza and GertrudHavliček for expert technical assistance, Dr. Beate Rinner andAlexandra Novak for helping with flow cytometric DNA measure-ments, Dr. Adelheid Kresse for some good advice regardingimmunofluorescence assays and Maria Teresa Botello Mulet forimproving the English text.

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α1-adrenergic drugs modulate differentiation and cell death of human erythroleukemia cells through...

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