Growth inhibition of mammalian cells by eosinophil cationic protein

Growth inhibition of mammalian cells by eosinophil cationic protein

Takashi Maeda1, Midori Kitazoe1, Hiroko Tada1, Rafael de Llorens2, David S. Salomon3,Masakazu Ueda4, Hidenori Yamada1 and Masaharu Seno1

1Department of Bioscience and Biotechnology, Faculty of Engineering, Graduate School of Natural Science and Technology,

Okayama University, Japan; 2Department of Biology, Faculty of Sciences, University of Girona, Spain;3Tumor Growth Factor Section, Basic Research Laboratory, National Cancer Institute, National Institutes of Health,

Bethesda, MD, USA; 4Department of Surgery, Keio University School of Medicine, Tokyo, Japan

Eosinophil cationic protein (ECP), one of the majorcomponents of basic granules of eosinophils, is cytotoxicto tracheal epithelium. However, the extent of this e�ecton other cell types has not been evaluated in vitro. In thisstudy, we evaluated the e�ect of ECP on 13 mammaliancell lines. ECP inhibited the growth of several cell linesincluding those derived from carcinoma and leukemia in adose-dependent manner. The IC50 values on A431 cells,MDA-MB-453 cells, HL-60 cells and K562 cells were esti-mated to be � 1±5 lM. ECP signi®cantly suppressed thesize of colonies of A431 cells, and decreased K562 cells inG1/G0 phase. However, there was little evidence that ECPkilled cells in either cell line. These e�ects of ECP were notenhanced by extending its N-terminus. Rhodamine B

isothiocyanate-labeled ECP started to bind to A431 cellsafter 0.5 h and accumulated for up to 24 h, indicating thatspeci®c a�nity for the cell surface may be important. Thea�nity of ECP for heparin was assessed and found to bereduced when tryptophan residues, one of which is locatedat a position in the catalytic subsite of ribonuclease inECP, were modi®ed. The growth-inhibitory e�ect was alsoattenuated by this modi®cation. These results suggest thatgrowth inhibition by ECP is dependent on cell type and iscytostatic.

Keywords: cell cycle; colony formation; cytostatic e�ect;eosinophil cationic protein (ECP); growth inhibition.

Eosinophil cationic protein (ECP) is one of the majorcomponents of eosinophilic granules with a molecular massranging from 16 to 21.4 kDa. It exhibits various biologicaleffects both in vitro and in vivo [1,2]. It is classi®ed as amember of the ribonuclease (RNase) A supergene familybecause of homology of both nucleotide and amino-acidsequences. The homology of amino-acid sequences betweenhuman ECP and human RNase 1 is � 30% [3,4]. On theother hand, ECP shows signi®cant sequence homology(70%) with eosinophil-derived neurotoxin (EDN), which isanother human RNase and a component of basic granulesin eosinophils [5]. Recently the 3D structure of ECP hasbeen determined and con®rmed the similarity of its structureto other members of the pancreatic-type RNases [6,7]. Somesubstitutions of amino-acid residues in the catalytic subsitesare consistent with the weak RNase activity of ECP. ECP is� 100±2000-fold less active than EDN depending on thetype of substrate [8,9].

ECP is bactericidal [10], helminthotoxic [11±17], elicits theGordon phenomenon when injected intrathecally into

rabbits [18,19], and is cytotoxic to tracheal epithelium[20,21]. Although the mechanism of its cytotoxicity is notcompletely understood, it is suggested to be due to the pore-forming activity of ECP,which destabilizes lipidmembranes[22] and is unrelated to its RNase activity [14,23]. This isconsistent with data showing that the cytotoxicity of ECP isgreater than that of EDN [13,19,24].

In this study, we have assessed the effect of ECP on thegrowth of 13 mammalian cell lines. The results show thatECP is growth inhibitory depending on the cell type and iscytostatic but not cytotoxic. Fluorescent labeled ECP isshown to enter the cell whereas RNase A does not. Aspeci®c af®nity for the cell surface may be part of itscytostatic effect. This ability of ECP to bind to the cellsurface is also shown to depend on tryptophan residues.

M A T E R I A L S A N D M E T H O D S

Cell cultures

Rat aortic smooth muscle A10 cells, human epidermoidcarcinoma A431 cells, squamous carcinoma TE-8 cellsderived from human esophageal cancer, HC-11 cells clonedfrom normal mouse mammary gland epithelia, andmouse metastatic melanoma-derived B16-BL6 cells weremaintained as described previously [25±27]. Simian virus(SV)-40-transformed Balb/c 3T3 cell line SV-T2 [28], SV-40transformed mouse Swiss/3T3 ®broblast cell line 3T3-SV40[29],mouse cell lineLL/2 established asLewis lung carcinoma[30], human colorectal adenocarcinoma cell line HT-29 [31],human chronic myelogenous leukemia cell line K562 [32],human acute promyelocytic leukemia cell line HL-60 [33],and human breast cancer cell lines MDA-MB-453 [34] and

Correspondence toM. Seno, Department of Bioscience and Biotech-

nology, Faculty of Engineering, Graduate school of Natural Science

and Technology, Okayama University, 3.1.1 Tsushima-Naka,

Okayama 700-8530, Japan. Fax/Tel.: +81 86 251 8216,

E-mail: [email protected]

Abbreviations: ECP, eosinophil cationic protein; EDN, eosinophil-

derived neurotoxin; RNase, ribonuclease; SV, simian virus; MTT,

3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide; RITC,

rhodamine B isothiocyanate; NBS, N-bromosuccinimide.

(Received 31 August 2001, revised 26 October 2001, accepted 5

November 2001)

Eur. J. Biochem. 269, 307±316 (2002) Ó FEBS 2002

T-47D [35] were obtained from American Type CultureCollection (USA)orDainippon-PharmaceuticalCo. (Japan)and maintained as directed.

Preparation of recombinant human ECP

Human ECP cDNA was isolated and expressed in anEscherichia coli T7 expression system as described previ-ously [7]. Computer analyses for the secretion signal [36,37]predicted the cleavage site of ECP to be between Gly23 andSer24. On the other hand, ECP puri®ed from normalhuman eosinophils had Arg28 at its N-terminus [38]. Hencetwo different types of ECP were prepared. To distinguishbetween them, the one with an N-terminal extension fromSer24 was designated (ÿ4) ECP. Puri®ed ECP and(ÿ4) ECP were assessed for RNase activity on yeast RNAby the perchloric acid precipitation method as previouslydescribed [39] and for bactericidal activity against Staphy-lococcus aureus 209P FDA by counting the colonies onplates [23]. The N-terminal sequences, CD spectra, andapparent molecular masses in SDS/PAGE of both proteinswere con®rmed to be as designed except for the ®rstmethionine residue of (ÿ4) ECP, which was processed off.

MTT assay for cell growth

The effect of ECP on the growth of various cell lines wasassessed by colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-diphenyl-tetrazolium bromide (MTT) [25]. Cells wereplated into 96-well plates (Nalge-Nunc, USA) in appropri-atemedia containing 10% fetal bovine serum at 500 cells perwell. After 24 h, each sample was added at the indicatedconcentration (0±10 lM). Four days after plating, themedium was replaced with fresh medium containing eachsample at the same concentration. After a further 4 days ofcultivation, MTT (5 mg�mLÿ1 in NaCl/Pi) was added, andcell growth was monitored by measuring A570.

Counting of viable cells

The number of K562 cells under various conditions wascounted. First, 25 000 cells were seeded into a 35-mm dish(Falcon), and appropriate concentrations of ECP (ÿ4) orECP and RNase A were simultaneously added. After1±3 days of cultivation, viable cells unstained with Trypanblue were counted with a hemocytometer.

Observation of cell morphology

K562 and A431 cells were seeded into 24-well plates or35-mm dishes, and, after 24 h, ECP was added at a concen-tration of 10 lM. At appropriate times during the culture,the morphology of the cells was observed with a phase-contrast inverted microscope (CK-2; Olympus, Tokyo,Japan) equippedwith a charge-coupled device video camera.

Cell cycle analysis

K562 cells were seeded at 500 000 cells per 60-mm dish inthe growthmedium.After 24 h, themediumwas changed tofreshmediumwith 10 lMECPor bovine RNase A (Sigma).After 3 days of treatment, cells were harvested, washed withNaCl/Pi, treated with NaCl/Pi containing 0.25% Triton

X-100 and 0.15 mg�mLÿ1 RNase A for 15 min at roomtemperature. The cells were then ®xed in 70% ethanolovernight at 4 °C and this was followed by furthertreatment with RNase A (0.1 mg�mLÿ1) in NaCl/Pi for10 min at 37 °C. The DNA of the ®xed cells was stainedwith propidium iodide (50 mg�mLÿ1) for 30 min at roomtemperature, and the cells were analyzed by FACSCalibur(Becton Dickinson).

Assay of colony formation

A431 cells suspended in the medium were seeded into35-mm dishes at 10 000 cells per dish. After 24 h, ECP wasadded at 10 lM to the medium and the cells were culturedfor an additional 3 days. The medium containing ECP wasthen changed. Seven days after seeding, the cells were ®xedwith 10% formaldehyde and stained with Crystal violet.The number of colonies was counted, and the area occupiedby the colonies was evaluated by image scanning assisted bya computer.

Fluorescence microscopy

ECP and bovine RNase A were labeled with rhodamine Bisothiocyanate (RITC; Sigma) as previously described [40].Cells were seeded into an eight-well Laboratory-TekChamber Slide (Nunc) at 20 000 cells per well and cultured.After 24 h, cells in each well were treated withRITC-labeledprotein at a concentration of 1 lM for 0.5±24 h, thenwashed with NaCl/Pi, and observed under a ¯uorescentmicroscope (BX40; Olympus). Hoechst 33342 dissolved inNaCl/Pi to 2 lM (Molecular Probes) was used for nuclearstaining.

Oxidation of tryptophan residues

Two tryptophan residues were modi®ed by oxidation withN-bromosuccinimide (NBS; Sigma) as previously described[41]. Brie¯y, 1.37 mg�mLÿ1 NBS dissolved in 50 mM

sodium acetate, pH 4.5, was gradually added to a solutionof ECP (1.6 mg�mLÿ1) in the same buffer. Oxidation wasmonitored by measuring the decrease in A280 during thecourse of the reaction. After dialysis against Milli-Q water,the solution of modi®ed ECP was assessed for amino-acidcomposition and presence of tryptophan residues.

Heparin-af®nity column chromatography

ECP with or without modi®cation was applied to a heparinaf®nity column (Heparin-Cellulo®ne; 4 ´ 150 mm; Chisso,Japan) equilibrated with 50 mM phosphate buffer, pH 7.0,and eluted with a linear gradient of NaCl (0.2±0.7 M per60 min) at a ¯ow rate of 0.6 mL�minÿ1. The A273 derivedfrom tyrosine residues was monitored and the af®nity ofeach protein for heparin was evaluated as the retention timeof the peak top of each pro®le.

R E S U L T S

ECPs with two different N-termini

As there is a discrepancy in the N-terminus between thepredicted and the puri®ed ECP protein, post-translational

308 T. Maeda et al. (Eur. J. Biochem. 269) Ó FEBS 2002

processing might be involved in the truncation of theN-terminal sequence of ECP. We thought that it wasimportant to assess the effect of this N-terminal extension inECP because EDN has a similar four amino-acid extendedform that conferred cytotoxic activity on KS Y-1 cells,which are neoplastic endothelial cells derived fromKaposi'ssarcoma [42]. We therefore expressed two types of recom-binant human ECP using the T7 expression system(Fig. 1A). RNase activity of ECP and (ÿ4) ECP againstyeast RNA was 100 times lower than that of bovineRNase A (Fig. 1B), which is consistent with a previousreport on the activity of ECP puri®ed from eosinophils [43].ECP and (ÿ4) ECP showed no difference in RNase activity(Fig. 1B). Both forms of ECP exhibited bactericidal activity

against S. aureus whereas RNase A did not show anyactivity (Fig. 1C). Therefore, both forms of recombinantECPs were biologically active.

Effect of ECPs on various cell lines

The growth-modulatory effects of ECP and (ÿ4) ECP wereassessed on 13 cell lines derived from humans and rodents.The results are summarized in Table 1. ECP showed thestrongest inhibition of growth in leukemia-derived cellsK562 and HL-60 with an IC50 (concentration that causes50% inhibition) of 1.1 lM. A431, MDA-MB-453 andHC-11 cells were also sensitive to ECP with IC50 values of� 4±6 lM. The (ÿ4) ECP protein also inhibited the growth

Fig. 1. N-Terminal sequences of ECP, its

ribonucleolytic activity and bactericidal activity.

(A) The signal cleavage site is predicted to lie

between Gly23 and Ser24 whereas the

N-terminus is reported to be Arg28 when

puri®ed from eosinophils. Recombinant ECP

was prepared as themature form starting from

Arg28 as indicated by the arrow at the top,

and (ÿ4) ECPwas prepared with an extension

of four amino-acid residues (in bold letters) as

also indicated by the arrow at the bottom.

RNase activity against yeast RNA (B) and

bactericidal activity against S. aureus (C) of

each ECP (d) and (ÿ4) ECP (j) were eval-

uated. Bovine RNase A (s) is a control.

Table 1. E�ect of ECP and (ÿ4) ECP on cell growth. All assays were carried out in quadruplicate in a 96-well plate and SD was calculated. SC,

Percentage of cells that survived at 10 lM ECP or (ÿ4) ECP; NT, not tested; NA, not applicable.

Cell line Origin

ECP (ÿ4) ECP

IC50

(lM)

SC

(%)

IC50

(lM)

SC

(%)

Human

K562 Chronic myelogenous leukemia 1.1 1.7 � 0.3 2.0 3.0 � 1.3

HL-60 Acute promyelocytic leukemia 1.1 2.1 � 0.5 2.0 5.4 � 2.4

A431 Epidermoid carcinoma 4.0 38.7 � 7.6 6.0 49.3 � 9.7

MDA-MB-453 Breast carcinoma (mammary gland) 4.0 31.3 � 4.7 NT NT

TE-8 Squamous carcinoma NA 68.5 � 2.8 NT NT

T-47D Ductal carcinoma (mammary gland) NA 90.3 � 3.9 NT NT

HT-29 Colon adenocarcinoma NA 75.3 � 4.7 NA 80.5 � 2.8

Mouse

B16-BL6 Metastatic melanoma NA 73.5 + 9.0 NT NT

LL/2 Lewis lung carcinoma NA 93.5 � 5.5 NA 100.6 � 4.5

3T3-SV40 SV-40-transformed Swiss 3T3 cells NA 57.6 � 9.8 NA 58.9 � 8.5

SV-T2 SV-40-transformed Balb/c 3T3 cells NA 90.3 � 8.7 NA 95.2 � 8.2

HC-11 Normal mammary gland epithelial cells 6.0 42.8 � 5.2 6.0 38.3 + 4.4

Rat

A10 Normal aortic smooth muscle cells NA 84.5 + 2.3 NT NT

Ó FEBS 2002 Cytostatic e�ect of ECP (Eur. J. Biochem. 269) 309

of these cell lines. However (ÿ4) ECP was less active thanECP. T-47D, LL/2 and SV-T2 cells were resistant to ECPwhile TE-8, HT-29, B16-BL6, 3T3-SV40 and A10 cells weremarginally sensitive such that the IC50 values could not beaccurately calculated but were more than 10 lM.

Effect of ECP on A431 and K562 cells

At 5 lM ECP, the growth of K562 cells was completelysuppressed whereas growth inhibition of A431 cells was� 50%(Fig. 2).AsECP is amember of the supergene familyof pancreatic-type RNases and is unique in this family forits basic pI, its growth-inhibitory effect was compared withthat of both bovine RNase A and poly(L-lysine) (averagemolecular mass � 2900). Neither RNase A nor poly(L-lysine) had any effect on the growth of these cells (Fig. 2).

After 7 days of incubation, ECP and (ÿ4) ECP appeared toinhibit the aggregation ofK562 cells and to keep them sparse(Fig 2C,D) in contrast with control cells or cells treated withRNase A, which grew as aggregates (Fig. 2A,B). It isinteresting to note that even (ÿ4) ECP, which allowed cellgrowth because of its weaker effect (Fig. 3A), appeared tosuppress cell aggregation. A431 cells are epithelial-like cellsand have a typical cobblestone appearance (Fig. 2E). Theyshow this cobblestone pattern even when seeded at lowerdensity, as shown in Fig. 4A for instance. Although thismorphology was not affected by RNase A (Fig. 2F), thecells treated with ECP for 5 days were more stellate inappearance (Fig. 2G). These cells resumed growth whenECP was removed from the culture medium (Fig. 2H).

As the growth inhibition of K562 cells in Fig. 2 wasshown after 7 days of treatment with ECP, which was

Fig. 2. Suppression of cell growth in the presence of ECP. Left, the percentages of viable cells under various concentrations of RNase A (s), poly

(L-lysine) (n), (ÿ4) ECP (j) and ECP (d) were plotted. Growth of K562 andA431 cells was monitored byMTT assay. Each assay was carried out

in quadruplicate and standard deviation was calculated and depicted in each vertical line. Right, K562 cells seeded at 500 cells per 35-mm dish were

cultured for 7 days in the regular medium (A) and in the presence of 10 lM eachRNase A (B), ECP (C) and (ÿ4) ECP (D). A431 cells seeded at 500

cells per 35-mm dish and cultured for 5 days in the regular medium (E) and in the presence of 10 lM each RNase A (F) and ECP (G). Four days

after plating, the medium was replaced with fresh medium containing each sample at the same concentration. ECP-treated A431 cells (G) were

further cultured for 3 days in the regularmediumwithout ECP (H). (G) and (H) show the same ®eld of the same dish. Original magni®cations of the

plates are ´ 10.


supplemented by the medium change, this effect wasassessed in the ®rst 3 days of treatment by monitoring thecell number (Fig. 3A). (ÿ4) ECPwas less active as a growthinhibitor during this period and the effect of ECP lastedalmost throughout not allowing any appreciable increase incell number. K562 cells treated with ECP were furtheranalyzed by ¯ow cytometry; a signi®cant decrease wasobserved in the population of cells in the G1/G0 phase of thecell cycle, and a small increase in the dying population whencompared with K562 cells cultured in regular medium orcells treated with RNase A (Fig. 3B). However, the popu-lation in the G2/M phase of the cell cycle was not altered,and the total cell number was unaffected. A small number ofK562 cells in the G1/G0 phase of the cell cycle that had beentreated with ECP or (ÿ4) ECP appeared to be dead.

The effect of ECP in the early period without mediumchange was monitored on A431 cells (Fig. 4). Up to 4 days

after the addition of ECP, A431 cells were more sparse andwere more ®broblastic in appearance (Figs 4D,E) in con-trast with control A431 cells (Fig. 4A,B) or A431 cellstreated with RNase A (Fig 4G,H). In the presence of ECP,the cells were more ¯at and spread out after 6 days oftreatment (Fig. 4F). Nuclei were more pronounced becauseof the low density and ¯attened shape of the cells (Figs 4A±C). Very recently, we found that ECP-treated Balb/c 3T3cells exhibited a similar change in morphology withenhanced expression of vinculin (M. Kitazoe, T. Maeda,H. Tada, R. de Llorens, D. S. Salomon, M. Ueda,H. Yamada & M. Seno, unpublished results). The effectof ECP on the cell shape might be due to the regulation ofvinculin gene expression as previously described [44,45].

The numbers of A431 colonies in the dishes (2500 � 110colonies per 35-mm dish) that received ECP or RNase Awere almost equivalent to the number of colonies in controlA431 cells (Fig. 5A). However, ECP produced a signi®cantdecrease in the size of the colonies of 60% compared withcontrol cells (Fig. 5B). These results demonstrate that ECPimpairs the growth of cells.

Cellular localization of ECP

To assess whether ECP could be localized, A431 cells wereincubated with RITC-labeled ECP at 37 °C for varioustimes (Fig. 6). From 0.5 to 3 h, A431 cells exhibitedincreased levels of ¯uorescence labeling in the cytoplasmrather than the nuclei. After 24 h, the ¯uorescence increasedin the cells. There was no uptake of RITC-labeled RNase Ainto the cells, indicating that ECP may interact preferen-tially with a receptor or binding protein on the cell surface.We attempted to observe the speci®c binding using ECP andRITC-labeled ECP by monitoring the level of ¯uorescence,but the change in ¯uorescence level caused by the compe-tition of ECP andRITC-labeled ECP could not be detected.This is probably because at least 1 lM RITC-labeled ECPwas required to detect the ¯uorescence, and this concentra-tionmay be too high to compete with the ECP, the practicalmaximum concentration of which is 10 lM. Although wecould not show speci®c binding of ECP to cells using acompetition assay, we could assess the af®nity of ECP forheparin using heparin af®nity column chromatography(Fig. 7A). ECP was eluted at about 0.64 M NaCl, and(ÿ4) ECP and NBS-modi®ed ECP were eluted at 0.60 and0.56 M NaCl, respectively. The amino-acid composition ofNBS-modi®ed ECP con®rmed that only tryptophan resi-dues had been modi®ed. As shown in Fig. 7B, thesetryptophan residues are located in the RNA catalytic siteof ECP and may contribute to the binding to heparin. Thecleft of the catalytic site possibly functions as the site ofattachment to proteoglycans on the cell surface. As NBS-modi®ed ECP inhibited the growth of A431 cells less than(ÿ4) ECP and the IC50 could not be determined (T.Maeda,D. L. Newton, S. M. Rybak, unpublished results), theaf®nity for heparin must also be responsible for the growth-inhibitory effect.

D I S C U S S I O N

This is the ®rst report to demonstrate that ECP has agrowth-inhibitory effect that is cytostatic and dependent oncell type. The growth of four of the seven human cell lines

Fig. 3. Growth-inhibitory e�ect of ECP on K562 cells. (A) Cells were

seeded at 25 000 cells per 35-mm dish. Simultaneously, 10 lM each

RNase A, ECP and (ÿ4) ECP were added to the medium. After the

indicated number of days of culture, the viable cells in the dishes were

counted. The cell numbers are the average from three independent

experiments and standard deviations are depicted by vertical lines on

the top of each bar. The horizontal gray line shows the cell number

seeded at the beginning of the experiments. (B) Cells were seeded at

500 000 cells per 60-mm dish, cultured for 3 days in the presence or

absence of 10 lM ECP or RNase A and analyzed by a ¯ow cytometer.

The area of dead cells is shaded. Peaks I and II show the population of

cells in G1/G0 and G2/M phase, respectively.


Fig. 4. Time course change in the morphology of A431 cells treated with ECP.A431 cells were seeded into a 24-well plate at 5000 cells per well. After

24 h, medium was changed to a fresh one (A, B, C) or one containing 10 lM ECP (D, E, F) or RNase A. (G, H, I). Cells were photographed 1 day

(A, D, G), 4 days (B, E, H) and 6 days (C, F, I) after the change of medium. Original magni®cations are ´ 10.

Fig. 5. Growth-inhibitory e�ect of ECP on

A431 cells.A431 cells were seeded at 5000 cells

per 35-mm dish, and 2500 � 110 colonies

were formed in the presence or absence of

10 lM RNase A or ECP (A). The percentage

of areas of colonies treated with RNase A and

ECP were calculated taking colonies cultured

in the growth medium as 100% (B). This

experiment was repeated three times. Photo-

graphs are the typical pattern of the colonies,

and the percentages are the means of each

result with the standard deviations within

10%.


was inhibited, whereas the rodent cell lines were relativelyresistant. The resistance of the rodent cell lines to thegrowth-inhibitory effects of human ECP may be due to theevolutionary divergence of ECP, which resulted in signi®-cantly low homology of eosinophil-derived RNase betweenspecies [46,47]. In this study, ECP suppressed the growth ofK562, HL-60, A431 and MDA-MB-453 cell lines in anIC50 range of 1±4 lM. Although the primary structure ofhuman ECP shows the closest identity (67%) with human

EDN, the N-terminal extension of which confers cytotox-icity against KS Y-1 cells [42], the N-terminal extensionof ECP did not produce any enhancement of the growth-inhibitory effects on this cell line (personal communi-cation, D. L. Newton and S. M. Rybak, National CancerInstitute, National Institutes of Health, Frederick, MD,USA). On the contrary, the N-terminal extension of ECPappears to impair the inhibitory effect of ECP on some celllines.

Fig. 6. A431 cells treated with RITC-labeled ECP.A431 cells were seeded into the eight-well Laboratory-Tek chamber slide at 20 000 cells per well.

After 24 h, 1 lM each RITC-labeled ECP and RNase A was added to the culture medium, and the cells were ®xed and detected by ¯uorescent

microscopy at the time indicated. RITC-labeled ECP or RNase A was visualized (top) and nuclei of the cells stained with Hoechst 33342 (bottom).

The same ®eld was assigned in the same column at each time. The scale bar is equivalent to 50 lm.

Fig. 7. Heparin a�nity column chromatography of ECP (A) and schematic diagram of ECP depicted with RasMol v2.6 according to the PDB entry

1DVT (B). (A) ECPmodi®edwithNBS (a) (ÿ4) ECP (b) or ECP (c) was applied to a heparin column and eluted with a linear gradient ofNaCl. The

retention time of each peak of pro®les is 43 min (0.64 M NaCl) for NBS-modi®ed ECP, 48 min (0.60 M NaCl) for (ÿ4) ECP and 53 min (0.56 M

NaCl) for ECP. RNase A passed through this a�nity column under the same conditions. (B) The backbone of peptide bonds is drawn in gray. The

secondary-structure elements are helices and arrows for ahelices and b strands, respectively. Two tryptophan residues,W10 andW35, and the other

amino-acid residues in the catalytic subsites are in blackwith their side chains. H64 is located in the P0 subsite, H15, K38 andH128 in the P1 subsite,

and W10 in the P2 subsite.


ECP is unique among RNase A enzymes because of itshigh arginine content. Therefore, the nonspeci®c effect ofgrowth inhibition may be replicated by other polycations.To investigate this, we checked the effect of poly(L-lysine),which has a cationic charge that is almost equivalent to thatof ECP. It did not have any growth-inhibitory effect onK562 andA431 cells (Fig. 2). Althoughwe could not obtainpolyarginine, we assessed the effect of the third helix of theDrosophila antennapedia homeodomain [48] and the basicregion of HIV-Tat protein [49], both of which are rich inbasic amino acids and known to enhance cellular uptake.Neither of these peptides showed any growth-inhibitoryeffect similar to ECP (data not shown). Therefore, thecationic charge could not replicate the effect of ECP.Recently, it has been shown that the amphipathic helixstructure of the basic region of Tat protein may beimportant for the uptake of the protein into cells [50]. Thismechanism may not apply to ECP as ECP is an arginine-rich protein but does not have the cluster of arginineresidues like Tat protein. On the other hand, highlycationized RNase molecules are cytotoxic but are notcytostatic like ECP [51].

The cytostatic growth-inhibitory effect of ECP was notsuf®cient to induce cell death, as the initial number of viableK562 cells did not signi®cantly decrease in the presence ofECP. Cell cycle analysis showed that ECP decreased K562cells in the G1/G0 phase without having an effect on the G2/M phase. The number of dying cells increased slightly butthis was not signi®cant. The growth-inhibitory effect of ECPwas reversible as A431 cells treated with ECP resumednormal growth when ECP was removed from the medium.In addition, ECP affected the size of colonies of A431 cellsbut not colony number. Furthermore, we found no effect ofECP on the frequency of apoptosis as assessed by DNAladder formation and phosphatidylserine externalization(data not shown).

RITC-labeled ECP appeared to accumulate in the cyto-plasm after 24 h. However, whether endocytosis of ECPocurred is still unclear. Itmaybe invesicles or endosomes.Asthe cellular outlines were not enhanced during the course ofthe incubation, we concluded that speci®c accumulation ofECPonthecell surfacewasnotoccurring.Themostprobableexplanation for this observation is the presence of cell sur-face receptors for ECP.We could not show the presence of aspeci®c high-af®nity binding site for ECP on cells by com-petition assay. This is probably because the af®nity of ECPfor binding to extracellular matrix proteins such as heparansulfate is extremely low and rapidly dissociates. However, itis very interesting to note that ECP can bind to heparin andthat tryptophan residues contribute to this af®nity. As tryp-tophan residues are also responsible for the af®nity forgalactoseand lactose residuesof some lectins [52], it is feasiblethat this is also the case for ECP. The tryptophan residuesare Trp10 and/or Trp35 in ECP. Interestingly, Trp10 islocated at the P2 subsite of the catalytic domain of RNaseand controls the weak RNase activity of the protein [6].Trp35 is a unique amino-acid residue at a position inLoop-3 of the RNases. As both tryptophan residues arelocated on the same side of the molecule, it is dif®cult toascertain which residue is more critical for the the binding ofECP to heparin or other carbohydrates.

In conclusion, ECP has a cell-type-speci®c cytostaticgrowth-inhibitory effect. It is possible that this activity is due

to the presence of the tryptophan residues. We are nowproducing mutant ECP proteins to assess this aspect.Further evaluation of the ECP molecule including thegeneration of a mutant protein will enable us to test theeffects of ECP on in¯ammatory diseases, in which uncon-trolled cell growth could contribute to a delay in woundhealing. In addition, strong local in¯ammatory responsesthat are speci®c to cytokines such as interleukin-4, arecapable in some cases of mediating regression of tumors[53,54]. In¯ammatory in®ltrates comprised of eosinophilsmay also play an important role at the primary site of tumorregression by releasing ECP as part of the cascade ofinducing a tumor-speci®c T-cell response. Optimization ofthe biological activity of the ECP protein to target the cellsurface could bring IC50 values down to practical levels sothat it might be used as an anti-cancer reagent in the sameway as others [27,55±57].

A C K N O W L E D G E M E N T S

We thankDrs S. Rybak andD. Newton for assaying the e�ects of ECP

and (ÿ4) ECP on KS Y-1 cells, Professor K. Oguma for providing

S. aureus cells, Professor M. Hikida for help with ¯ow-cytometric

analysis, and Drs R. Sasada, S. Ishikami and J. Futami for helpful

discussions and suggestions throughout this work. This workwas partly

supported by a grant-in-aid from the Ministry of Education, Science,

Culture and Sports of Japan. R. de Ll. was supported by the Spanish

Ministerio de EducacioÂ n y Cultura (grant SAF 98-0086 and 2FD97-

0872) and by Generalitat de Catalunya (grant SGR97-240).

R E F E R E N C E S

1. Rosenberg, H.F. (1998) The eosinophil ribonucleases. Cell. Mol.

Life Sci. 54, 795±803.

2. Giembycz, M.A. & Lindsay, M.A. (1999) Pharmacology of the

eosinophil. Pharmacol. Rev. 51, 213±340.

3. Seno, M., Futami, J., Kosaka, M., Seno, S. & Yamada, H. (1994)

Nucleotide sequence encoding human pancreatic ribonuclease.

Biochim. Biophys. Acta 1218, 466±468.

4. Futami, J., Tsushima, Y., Murato, Y., Tada, H., Sasaki, J.,

Seno, M. & Yamada, H. (1997) Tissue-speci®c expression of

pancreatic-type RNases and RNase inhibitor in humans. DNA

Cell Biol. 16, 413±419.

5. Gleich, G.J., Loegering, D.A., Bell, M.P., Checkel, J.L., Acker-

man, S.J. & McKean, D.J. (1986) Biochemical and functional

similarities between human eosinophil-derived neurotoxin and

eosinophil cationic protein: homology with ribonuclease. Proc.

Natl Acad. Sci. USA 83, 3146±3150.

6. Boix, E., Leonidas, D.D., Nikolovski, Z., Nogues,M.V., Cuchillo,

C.M. & Acharya, K.R. (1999) Crystal structure of eosinophil

cationic protein at 2.4 AÊ resolution.Biochemistry 38, 16794±16801.

7. Mallorqui-Fernandez, G., Pous, J., Peracaula, R., Aymami, J.,

Maeda, T., Tada, H., Yamada, H., Seno, M., de Llorens, R.,

Gomis-Ruth, F.X. & Coll, M. (2000) Three-dimensional crystal

structure of human eosinophil cationic protein (RNase 3) at 1.75

AÊ resolution. J. Mol. Biol. 300, 1297±1307.

8. Slifman, N.R., Loegering, D.A., McKean, D.J. & Gleich, G.J.

(1986) Ribonuclease activity associated with human eosinophil-

derived neurotoxin and eosinophil cationic protein. J. Immunol.

137, 2913±2917.

9. Rosenberg, H.F. & Dyer, K.D. (1997) Diversity among the pri-

mate eosinophil-derived neurotoxin genes: a speci®c C-terminal

sequence is necessary for enhanced ribonuclease activity. Nucleic

Acids Res. 25, 3532±3536.

10. Lehrer, R.I., Szklarek, D., Barton, A., Ganz, T., Hamann, K.J. &

Gleich, G.J. (1989) Antibacterial properties of eosinophil major


basic protein and eosinophil cationic protein. J. Immunol. 142,

4428±4434.

11. Ackerman, S.J., Gleich, G.J., Loegering, D.A., Richardson, B.A.

& Butterworth, A.E. (1985) Comparative toxicity of puri®ed

human eosinophil granule cationic proteins for schistosomula

of Schistosoma mansoni. Am. J. Trop. Med. Hyg. 34, 735±745.

12. Hamann, K.J., Gleich, G.J., Checkel, J.L., Loegering, D.A.,

McCall, J.W. & Barker, R.L. (1990) In vitro killing of micro®lariae

of Brugia pahangi and Brugia malayi by eosinophil granule pro-

teins. J. Immunol. 144, 3166±3173.

13. Hamann, K.J., Barker, R.L., Loegering, D.A. & Gleich, G.J.

(1987) Comparative toxicity of puri®ed human eosinophil granule

proteins for newborn larvae of Trichinella spiralis. J. Parasitol. 73,

523±529.

14. Molina, H.A., Kierszenbaum, F., Hamann, K.J. & Gleich, G.J.

(1988) Toxic e�ects produced or mediated by human eosinophil

granule components on Trypanosoma cruzi. Am. J. Trop. Med.

Hyg. 38, 327±334.

15. McLaren, D.J., McKean, J.R., Olsson, I., Venges, P. & Kay, A.B.

(1981) Morphological studies on the killing of schistosomula of

Schistosomamansoni by human eosinophil and neutrophil cationic

proteins in vitro. Parasite Immunol. 3, 359±373.

16. Yazdanbakhsh, M., Tai, P.C., Spry, C.J., Gleich, G.J. & Roos, D.

(1987) Synergism between eosinophil cationic protein and oxygen

metabolites in killing of schistosomula of Schistosoma mansoni.

J. Immunol. 138, 3443±3447.

17. Waters, L.S., Taverne, J., Tai, P.C., Spry, C.J., Targett, G.A. &

Playfair, J.H. (1987) Killing of Plasmodium falciparum by eosin-

ophil secretory products. Infect. Immun. 55, 877±881.

18. Durack, D.T., Sumi, S.M. & Klebano�, S.J. (1979) Neurotoxicity

of human eosinophils. Proc. Natl Acad. Sci. USA 76, 1443±1447.

19. Fredens, K., Dahl, R. & Venge, P. (1982) The Gordon phenom-

enon induced by the eosinophil cationic protein and eosinophil

protein X. J. Allergy Clin. Immunol. 70, 361±366.

20. Motojima, S., Frigas, E., Loegering, D.A. & Gleich, G.J. (1989)

Toxicity of eosinophil cationic proteins for guinea pig tracheal

epithelium in vitro. Am. Rev. Respir. Dis. 139, 801±805.

21. Fredens, K., Dybdahl, H., Dahl, R. & Baandrup, U. (1988)

Extracellular deposit of the cationic proteins ECP and EPX in

tissue in®ltrations of eosinophils related to tissue damage.APMIS

96, 711±719.

22. Young, J.D., Peterson, C.G., Venge, P. & Cohn, Z.A. (1986)

Mechanism of membrane damage mediated by human eosinophil

cationic protein. Nature (London) 321, 613±616.

23. Rosenberg, H.F. (1995) Recombinant human eosinophil cationic

protein. Ribonuclease activity is not essential for cytotoxicity.

J. Biol. Chem. 270, 7876±7881.

24. Barker, R.L., Ten Loegering, D.A.R.M., Hamann, K.J., Pease,

L.R. & Gleich, G.J. (1989) Eosinophil cationic protein cDNA.

Comparison with other toxic cationic proteins and ribonucleases.

J. Immunol. 143, 952±955.

25. Seno, M., Tada, H., Kosaka, M., Sasada, R., Igarashi, K., Shing,

Y., Folkman, J., Ueda, M. & Yamada, H. (1996) Human beta-

cellulin, a member of the EGF family dominantly expressed in

pancreas and small intestine, is fully active in a monomeric form.

Growth Factors 13, 181±191.

26. Seno, M., DeSantis, M., Kannan, S., Bianco, C., Tada, H., Kim,

N., Kosaka, M., Gullick, W.J., Yamada, H. & Salomon, D.S.

(1998) Puri®cation and characterization of a recombinant human

cripto-1 protein. Growth Factors 15, 215±229.

27. Futami, J., Seno, M., Ueda, M., Tada, H. & Yamada, H. (1999)

Inhibition of cell growth by a fused protein of human ribonuclease

1 and human basic ®broblast growth factor. Protein Eng. 12,

1013±1019.

28. Aaronson, S.A. & Todaro, G.J. (1968) Development of 3T3-like

lines from Balb-c mouse embryo cultures: transformation sus-

ceptibility to SV40. J. Cell. Physiol. 72, 141±148.

29. O'Neill, F.J. (1975) Control of nuclear division in sv40 and ade-

novirus type 12 transformed mouse 3t3 cells. Int. J. Cancer 15,

715±723.

30. Bertram, J.S. & Janik, P. (1980) Establishment of a cloned line of

Lewis Lung Carcinoma cells adapted to cell culture. Cancer Lett.

11, 63±73.

31. von Kleist, S., Chany, E., Burtin, P., King, M. & Fogh, J. (1975)

Immunohistology of the antigenic pattern of a continuous

cell line from a human colon tumor. J. Natl Cancer Inst. 55,

555±560.

32. Andersson, L.C., Nilsson, K. & Gahmberg, C.G. (1979) K562: a

human erythroleukemic cell line. Int. J. Cancer 23, 143±147.

33. Collins, S.J., Gallo, R.C. & Gallagher, R.E. (1977) Continuous

growth and di�erentiation of human myeloid leukaemic cells in

suspension culture. Nature (London) 270, 347±349.

34. Cailleau, R., Olive,M. &Cruciger, Q.V. (1978) Long-term human

breast carcinoma cell lines of metastatic origin: preliminary char-

acterization. In Vitro 14, 911±915.

35. Prager, A., Ben-Hur, E., Chaitcik, S., Brenner, H.J. & Riklis, E.

(1981) Characterization of the response of a human breast carci-

noma cell line (T-47D) to radiation and chemotherapeutic agents.

Isr. J. Med. Sci. 17, 976±979.

36. Nakai, K. & Kanehisa, M. (1992) A knowledge base for predict-

ing protein localization sites in eukaryotic cells. Genomics 14,

897±911.

37. Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. (1997)

Identi®cation of prokaryotic and eukaryotic signal peptides and

prediction of their cleavage sites. Protein Eng. 10, 1±6.

38. Peterson, C.G., Jornvall, H. & Venge, P. (1988) Puri®cation and

characterization of eosinophil cationic protein from normal

human eosinophils. Eur J. Haematol. 40, 415±423.

39. Futami, J., Seno, M., Kosaka, M., Tada, H., Seno, S. &

Yamada, H. (1995) Recombinant human pancreatic ribonuclease

produced in E. coli: importance of the amino-terminal sequence.

Biochem. Biophys. Res. Commun. 216, 406±413.

40. Hiratsuka, T. (1987) Selective ¯uorescent labeling of the 50-, 26-,

and 20-kilodalton heavy chain segments of myosin ATPase.

J. Biochem. (Tokyo) 101, 1457±1462.

41. Spande, T.F., Green, N.M. & Witkop, B. (1966) The reactivity

towardN-bromosuccinimide of tryptophan in enzymes, zymogens,

and inhibited enzymes. Biochemistry 5, 1926±1933.

42. Newton, D.L. & Rybak, S.M. (1998) Unique recombinant human

ribonuclease and inhibition of Kaposi's sarcoma cell growth.

J. Natl Cancer Inst. 90, 1787±1791.

43. Sorrentino, S. & Libonati, M. (1997) Structure±function rela-

tionships in human ribonucleases: main distinctive features of the

major RNase types. FEBS Lett. 404, 1±5.

44. Rodriguez Fernandez, J.L., Geiger, B., Salomon, D., Sabanay, I.,

Zoller, M. & Ben-Ze'ev, A. (1992) Suppression of tumorigenicity

in transformed cells after transfection with vinculin cDNA. J. Cell

Biol. 119, 427±438.

45. Rodriguez Fernandez, J.L., Geiger, B., Salomon, D. & Ben-Ze'ev,

A. (1993) Suppression of vinculin expression by antisense trans-

fection confers changes in cell morphology, motility, and ancho-

rage-dependent growth of 3T3 cells. J. Cell Biol. 122, 1285±1294.

46. Larson, K.A., Olson, E.V., Madden, B.J., Gleich, G.J., Lee, N.A.

& Lee, J.J. (1996) Two highly homologous ribonuclease genes

expressed in mouse eosinophils identify a larger subgroup of the

mammalian ribonuclease superfamily. Proc. Natl Acad. Sci. USA

93, 12370±12375.

47. Singhania, N.A., Dyer, K.D., Zhang, J., Deming, M.S., Bonville,

C.A., Domachowske, J.B. & Rosenberg, H.F. (1999) Rapid evo-

lution of the ribonuclease A superfamily: adaptive expansion of

independent gene clusters in rats and mice. J. Mol. Evol. 49, 721±

728.

48. Derossi, D., Calvet, S., Trembleau, A., Brunissen, A., Chassaing,

G. & Prochiantz, A. (1996) Cell internalization of the third helix of


the Antennapedia homeodomain is receptor-independent. J. Biol.

Chem. 271, 18188±18193.

49. Schwarze, S.R., Ho, A., Vocero-Akbani, A. &Dowdy, S.F. (1999)

In vivo protein transduction: delivery of a biologically active

protein into the mouse. Science 285, 1569±1572.

50. Ho, A., Schwarze, S.R., Mermelstein, S.J., Waksman, G. &

Dowdy, S.F. (2001) Synthetic protein transduction domains:

enhanced transduction potential in vitro and in vivo. Cancer Res.

61, 474±477.

51. Futami, J., Maeda, T., Kitazoe, M., Nukui, E., Tada, H.,

Seno, M., Kosaka, M. & Yamada, H. (2001) Preparation of

potent cytotoxic ribonucleases by cationization: enhanced cellular

uptake and decreased interaction with ribonuclease inhibitor

by chemical modi®cation of carboxyl groups. Biochemistry 40,

7518±7524.

52. Rini, J.M. (1995) Lectin structure. Annu. Rev. Biophys. Biomol.

Struct. 24, 551±577.

53. Tepper, R.I., Co�man, R.L. & Leder, P. (1992) An eosinophil-

dependent mechanism for the antitumor e�ect of interleukin-4.

Science 257, 548±551.

54. Pardoll, D.M. (1995) Paracrine cytokine adjuvants in cancer

immunotherapy. Annu. Rev. Immunol. 13, 399±415.

55. Psarras, K., Ueda, M., Yamamura, T., Ozawa, S., Kitajima, M.,

Aiso, S., Komatsu, S. & Seno, M. (1998) Human pancreatic

RNase1-human epidermal growth factor fusion: an entirely

human Ôimmunotoxin analogÕ with cytotoxic properties against

squamous cell carcinomas. Protein Eng. 11, 1285±1292.

56. Psarras, K., Ueda, M., Tanabe, M., Kitajima, M., Aiso, S.,

Komatsu, S. & Seno, M. (2000) Targeting activated lymphocytes

with an entirely human immunotoxin analogue: human pancreatic

RNase1-human IL-2 fusion. Cytokine 12, 786±790.

57. Reiter, Y. & Pastan, I. (1998) Recombinant Fv immunotoxins and

Fv fragments as novel agents for cancer therapy and diagnosis.

Trends Biotechnol. 16, 513±520.


Growth inhibition of mammalian cells by eosinophil cationic protein

Documents

Transcript of Growth inhibition of mammalian cells by eosinophil cationic protein