Báo cáo Y học: Growth inhibition of mammalian cells by eosinophil cationic protein pptx

Growth inhibition of mammalian cells by eosinophil cationic protein Takashi Maeda’, Midori Kitazoe', Hiroko Tada’, Rafael de Llorens”, David S.. Keywords: cell cycle; colony formation; c

Growth inhibition of mammalian cells by eosinophil cationic protein Takashi Maeda’, Midori Kitazoe', Hiroko Tada’, Rafael de Llorens”, David S Salomon’,

Masakazu Ueda‘, Hidenori Yamada' and Masaharu Seno!

‘Department of Bioscience and Biotechnology, Faculty of Engineering, Graduate School of Natural Science and Technology, Okayama University, Japan; ? Department 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; *Department of Surgery, Keio University School of Medicine, Tokyo, Japan

Eosinophil cationic protein (ECP), one of the major

components of basic granules of eosinophils, is cytotoxic

to tracheal epithelium However, the extent of this effect

on other cell types has not been evaluated in vitro In this

study, we evaluated the effect of ECP on 13 mammalian

cell lines ECP inhibited the growth of several cell lines

including those derived from carcinoma and leukemia in a

dose-dependent manner The ICs 9 values on A431 cells,

MDA-MB-453 cells, HL-60 cells and K562 cells were esti-

mated to be + 1-5 um ECP significantly suppressed the

size of colonies of A431 cells, and decreased K562 cells in

G,/Go phase However, there was little evidence that ECP

killed cells in either cell line These effects of ECP were not

enhanced by extending its N-terminus Rhodamine B

isothiocyanate-labeled ECP started to bind to A431 cells after 0.5 h and accumulated for up to 24 h, indicating that specific affinity for the cell surface may be important The affinity of ECP for heparin was assessed and found to be reduced when tryptophan residues, one of which is located

at a position in the catalytic subsite of ribonuclease in ECP, were modified The growth-inhibitory effect was also attenuated by this modification These results suggest that growth inhibition by ECP is dependent on cell type and is cytostatic

Keywords: cell cycle; colony formation; cytostatic effect; eosinophil cationic protein (ECP); growth inhibition

Eosinophil cationic protein (ECP) is one of the major

components of eosinophilic granules with a molecular mass

ranging from 16 to 21.4 kDa It exhibits various biological

effects both im vitro and in vivo [1,2] It is classified as a

member of the ribonuclease (RNase) A supergene family

because of homology of both nucleotide and amino-acid

sequences The homology of amino-acid sequences between

human ECP and human RNase | is © 30% [3,4] On the

other hand, ECP shows significant sequence homology

(70%) with eosinophil-derived neurotoxin (EDN), which is

another human RNase and a component of basic granules

in eosinophils [5] Recently the 3D structure of ECP has

been determined and confirmed the similarity of its structure

to other members of the pancreatic-type RNases [6,7] Some

substitutions of amino-acid residues in the catalytic subsites

are consistent with the weak RNase activity of ECP ECP is

=~ 100—2000-fold less active than EDN depending on the

type of substrate [8,9]

ECP is bactericidal [10], helminthotoxic [11-17], elicits the

Gordon phenomenon when injected intrathecally into

Correspondence to M 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: ssnom@biotech.okayama-u.ac.jp

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)

rabbits [18,19], and is cytotoxic to tracheal epithelium [20,21] Although the mechanism of its cytotoxicity is not completely understood, it is suggested to be due to the pore- forming activity of ECP, which destabilizes lipid membranes [22] and is unrelated to its RNase activity [14,23] This is consistent with data showing that the cytotoxicity of ECP is greater than that of EDN [13,19,24]

In this study, we have assessed the effect of ECP on the growth of 13 mammalian cell lines The results show that ECP is growth inhibitory depending on the cell type and is cytostatic but not cytotoxic Fluorescent labeled ECP is shown to enter the cell whereas RNase A does not A specific affinity for the cell surface may be part of its cytostatic effect This ability of ECP to bind to the cell surface is also shown to depend on tryptophan residues

MATERIALS AND METHODS Cell cultures

Rat aortic smooth muscle A10 cells, human epidermoid carcinoma A431 cells, squamous carcinoma TE-8 cells derived from human esophageal cancer, HC-11 cells cloned from normal mouse mammary gland epithelia, and mouse metastatic melanoma-derived B16-BL6 cells were maintained as described previously [25-27] Simian virus (SV)-40-transformed Balb/c 3T3 cell line SV-T2 [28], SV-40 transformed mouse Swiss/3T3 fibroblast cell line 3T3-SV40 [29], mouse cell line LL/2 established as Lewis 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

T-47D [35] were obtained from American Type Culture

Collection (USA) or Dainippon-Pharmaceutical Co (Japan)

and maintained as directed

Preparation of recombinant human ECP

Human ECP cDNA was isolated and expressed in an

Escherichia 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 and

Ser24 On the other hand, ECP purified from normal

human eosinophils had Arg28 at its N-terminus [38] Hence

two different types of ECP were prepared To distinguish

between them, the one with an N-terminal extension from

Ser24 was designated (—4) ECP Purified ECP and

(—4) ECP were assessed for RNase activity on yeast RNA

by the perchloric acid precipitation method as previously

described [39] and for bactericidal activity against Staphy-

lococcus aureus 209P FDA by counting the colonies on

plates [23] The N-terminal sequences, CD spectra, and

apparent molecular masses in SDS/PAGE of both proteins

were confirmed to be as designed except for the first

methionine residue of (—4) ECP, which was processed off

MTT assay for cell growth

The effect of ECP on the growth of various cell lines was

assessed by colorimetric assay using 3-(4,5-dimethylthiazol-

2-yl)-diphenyl-tetrazolium bromide (MTT) [25] Cells were

plated into 96-well plates (Nalge-Nunc, USA) in appropri-

ate media containing 10% fetal bovine serum at 500 cells per

well After 24 h, each sample was added at the indicated

concentration (0-10 um) Four days after plating, the

medium was replaced with fresh medium containing each

sample at the same concentration After a further 4 days of

cultivation, MTT (5 mg-mL~! in NaCl/P;) was added, and

cell growth was monitored by measuring 457

Counting of viable cells

The number of K562 cells under various conditions was

counted First, 25 000 cells were seeded into a 35-mm dish

(Falcon), and appropriate concentrations of ECP (—4) or

ECP and RNase A were simultaneously added After

1-3 days of cultivation, viable cells unstained with Trypan

blue were counted with a hemocytometer

Observation of cell morphology

K562 and A431 cells were seeded into 24-well plates or

35-mm dishes, and, after 24 h, ECP was added at a concen-

tration of 10 um At appropriate times during the culture,

the morphology of the cells was observed with a phase-

contrast inverted microscope (CK-2; Olympus, Tokyo,

Japan) equipped with a charge-coupled device video camera

Cell cycle analysis

K562 cells were seeded at 500 000 cells per 60-mm dish in

the growth medium After 24 h, the medium was changed to

fresh medium with 10 um ECP or bovine RNase A (Sigma)

After 3 days of treatment, cells were harvested, washed with

NaCl/P;, treated with NaCl/P; containing 0.25% Triton

X-100 and 0.15 mg-mL~!' RNase A for 15 min at room temperature The cells were then fixed in 70% ethanol overnight at 4°C and this was followed by further treatment with RNase A (0.1 mg-mL~') in NaCl/P; for

10 min at 37 °C The DNA of the fixed cells was stained with propidium iodide (50 mg-mL~') for 30 min at room temperature, and the cells were analyzed by FACSCalibur (Becton Dickinson)

Assay of colony formation A431 cells suspended in the medium were seeded into 35-mm dishes at 10 000 cells per dish After 24 h, ECP was added at 10 um to the medium and the cells were cultured for an additional 3 days The medium containing ECP was then changed Seven days after seeding, the cells were fixed with 10% formaldehyde and stained with Crystal violet The number of colonies was counted, and the area occupied

by the colonies was evaluated by image scanning assisted by

a computer

Fluorescence microscopy ECP and bovine RNase A were labeled with rhodamine B isothiocyanate (RITC; Sigma) as previously described [40] Cells were seeded into an eight-well Laboratory-Tek Chamber Slide (Nunc) at 20 000 cells per well and cultured After 24 h, cells in each well were treated with RITC-labeled protein at a concentration of | um for 0.5—-24h, then washed with NaCl/P;, and observed under a fluorescent microscope (BX40; Olympus) Hoechst 33342 dissolved in NaCl/P; to 2 um (Molecular Probes) was used for nuclear staining

Oxidation of tryptophan residues Two tryptophan residues were modified by oxidation with N-bromosuccinimide (NBS; Sigma) as previously described [41] Briefly, 1.37 mgmL~' NBS dissolved in 50 mm sodium acetate, pH 4.5, was gradually added to a solution

of ECP (1.6 mg:mL”) in the same buffer Oxidation was monitored by measuring the decrease in A>g9 during the course of the reaction After dialysis against Milli-Q water, the solution of modified ECP was assessed for amino-acid composition and presence of tryptophan residues

Heparin-affinity column chromatography ECP with or without modification was applied to a heparin affinity column (Heparin-Cellulofine; 4 x 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 per

60 min) at a flow rate of 0.6 mL-min | The A573 derived from tyrosine residues was monitored and the affinity of each protein for heparin was evaluated as the retention time

of the peak top of each profile

RESULTS ECPs with two different N-termini

As there is a discrepancy in the N-terminus between the predicted and the purified ECP protein, post-translational

A

ture ECP

mvpklftsqiclllllglmgvegSLHARPPOQFTR -

Fig 1 N-Terminal sequences of ECP, its 04 C 108

ribonucleolytic activity and bactericidal activity "

(A) The signal cleavage site is predicted to lie 03- 105

between Gly23 and Ser24 whereas the Ẹ ‘ §

purified from eosinophils Recombinant ECP 8 02-¬ 8

was prepared as the mature form starting from ® 5 > 10°

and (—4) ECP was prepared with an extension g 7 QB 10

of four amino-acid residues (in bold letters) as 3 0.0 = 40

also indicated by the arrow at the bottom

bactericidal activity against S aureus (C) of 0001 001 001 04 1 5 0.4 1 10 each ECP (@) and (—4) ECP (i) were eval-

Protein concentration (uM)

uated Bovine RNase A (©) is a control Protein concentration (1M)

processing might be involved in the truncation of the

N-terminal sequence of ECP We thought that it was

important to assess the effect of this N-terminal extension in

against S aureus whereas RNase A did not show any activity (Fig 1C) Therefore, both forms of recombinant ECPs were biologically active

ECP because EDN has a similar four amino-acid extended

form that conferred cytotoxic activity on KS Y-1 cells,

which are neoplastic endothelial cells derived from Kaposi’s

sarcoma [42] We therefore expressed two types of recom-

binant human ECP using the T7 expression system

(Fig 1A) RNase activity of ECP and (—4) ECP against

yeast RNA was 100 times lower than that of bovine

RNase A (Fig 1B), which is consistent with a previous

report on the activity of ECP purified from eosinophils [43]

ECP and (—4) ECP showed no difference in RNase activity

(Fig 1B) Both forms of ECP exhibited bactericidal activity

Effect of ECPs on various cell lines The growth-modulatory effects of ECP and (—4) ECP were assessed on 13 cell lines derived from humans and rodents The results are summarized in Table 1 ECP showed the strongest inhibition of growth in leukemia-derived cells K562 and HL-60 with an ICsp (concentration that causes 50% inhibition) of 1.1 wm A431, MDA-MB-453 and HC-11 cells were also sensitive to ECP with ICso values of

46 um The (—4) ECP protein also inhibited the growth

Table 1 Effect of ECP and (—4) 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 tum ECP or (—4) ECP; NT, not tested; NA, not applicable

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

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

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

Al0 Normal aortic smooth muscle cells NA 84.5 + 2.3 NT NT

of these cell lines However (—4) ECP was less active than

ECP T-47D, LL/2 and SV-T2 cells were resistant to ECP

while TE-8, HT-29, B16-BL6, 3T3-SV40 and A10 cells were

marginally sensitive such that the [C59 values could not be

accurately calculated but were more than 10 pm

Effect of ECP on A431 and K562 cells

At 5 um ECP, the growth of K562 cells was completely

suppressed whereas growth inhibition of A431 cells was

+ 50% (Fig 2) As ECP is a member of the supergene family

of pancreatic-type RNases and is unique in this family for

its basic pI, its growth-inhibitory effect was compared with

that of both bovine RNase A and poly(1-lysine) (average

molecular mass = 2900) Neither RNase A nor poly

(L-lysine) had any effect on the growth of these cells (Fig 2)

K562 |

=100,

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5 ö 75

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= 50

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6 25

S

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Protein concentration (uM)

A431

=100

50 -

After 7 days of incubation, ECP and (—4) ECP appeared to inhibit the aggregation of K 562 cells and to keep them sparse (Fig 2C,D) in contrast with control cells or cells treated with RNase A, which grew as aggregates (Fig 2A,B) It is interesting to note that even (—4) ECP, which allowed cell growth because of its weaker effect (Fig 3A), appeared to suppress cell aggregation A431 cells are epithelial-like cells and have a typical cobblestone appearance (Fig 2E) They show this cobblestone pattern even when seeded at lower density, as shown in Fig 4A for instance Although this morphology was not affected by RNase A (Fig 2F), the cells treated with ECP for 5 days were more stellate in appearance (Fig 2G) These cells resumed growth when ECP was removed from the culture medium (Fig 2H)

As the growth inhibition of K562 cells in Fig 2 was shown 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 (©), poly (L-lysine) (A), (—4) ECP (Ml) and ECP (@) were plotted Growth of K562 and A431 cells was monitored by MTT assay Each assay was carried out

in quadruplicate and standard deviation was calculated and depicted in each vertical line Right, K 562 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 jum each RNase A (B), ECP (C) and (—4) 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 tm 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 regular medium without ECP (H) (G) and (H) show the same field of the same dish Original magnifications of the plates are x 10.

A

30 :

N ol

° Medium RNase A

I I —] II I II

number 60

0 200 400 600 0 200 400 600 0 200 400 600

DNA Content

Fig 3 Growth-inhibitory effect of ECP on K562 cells (A) Cells were

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

RNase A, ECP and (—4) 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 um ECP or RNase A and analyzed by a flow cytometer

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

cells in G,/Gop and G>/M phase, respectively

supplemented by the medium change, this effect was

assessed In the first 3 days of treatment by monitoring the

cell number (Fig 3A) (—4) ECP was less active as a growth

inhibitor during this period and the effect of ECP lasted

almost throughout not allowing any appreciable increase in

cell number K562 cells treated with ECP were further

analyzed by flow cytometry; a significant decrease was

observed 1n the population of cells in the G,/Gp phase of the

cell cycle, and a small increase 1n the dying population when

compared with K562 cells cultured in regular medium or

cells treated with RNase A (Fig 3B) However, the popu-

lation in the G>/M phase of the cell cycle was not altered,

and the total cell number was unaffected A small number of

K 562 cells in the G,/Gp phase of the cell cycle that had been

treated with ECP or (—4) ECP appeared to be dead

The effect of ECP in the early period without medium

change was monitored on A431 cells (Fig 4) Up to 4 days

after the addition of ECP, A431 cells were more sparse and were more fibroblastic in appearance (Figs 4D,E) in con- trast with control A431 cells (Fig 4A,B) or A431 cells treated with RNase A (Fig 4G,H) In the presence of ECP, the cells were more flat and spread out after 6 days of treatment (Fig 4F) Nuclei were more pronounced because

of the low density and flattened shape of the cells (Figs 4A— C) Very recently, we found that ECP-treated Balb/c 3T3 cells exhibited a similar change in morphology with

enhanced expression of vinculin (M Kitazoe, T Maeda,

H Tada, R de Llorens, D S Salomon, M Ueda,

H Yamada & M Seno, unpublished results) The effect

of ECP on the cell shape might be due to the regulation of vinculin gene expression as previously described [44,45] The numbers of A431 colonies in the dishes (2500 + 110 colonies per 35-mm dish) that received ECP or RNase A were almost equivalent to the number of colonies in control A431 cells (Fig SA) However, ECP produced a significant decrease 1n the size of the colonies of 60% compared with control cells (Fig 5B) These results demonstrate that ECP impairs the growth of cells

Cellular localization of ECP

To assess whether ECP could be localized, A431 cells were

incubated with RITC-labeled ECP at 37 °C for various times (Fig 6) From 0.5 to 3h, A431 cells exhibited increased levels of fluorescence labeling in the cytoplasm

rather than the nuclei After 24 h, the fluorescence increased

in the cells There was no uptake of RITC-labeled RNase A into the cells, indicating that ECP may interact preferen- tially with a receptor or binding protein on the cell surface

We attempted to observe the specific binding using ECP and RITC-labeled ECP by monitoring the level of fluorescence, but the change in fluorescence level caused by the compe- tition of ECP and RITC-labeled ECP could not be detected This is probably because at least 1 um RITC-labeled ECP was required to detect the fluorescence, and this concentra- tion may be too high to compete with the ECP, the practical maximum concentration of which is 10 um Although we could not show specific binding of ECP to cells using a competition assay, we could assess the affinity of ECP for heparin using heparin affinity column chromatography (Fig 7A) ECP was eluted at about 0.64 mM NaCl, and (—4) ECP and NBS-modified ECP were eluted at 0.60 and 0.56 M NaCl, respectively The amino-acid composition of NBS-modified ECP confirmed that only tryptophan resi- dues had been modified As shown in Fig 7B, these tryptophan residues are located in the RNA catalytic site

of ECP and may contribute to the binding to heparin The cleft of the catalytic site possibly functions as the site of attachment to proteoglycans on the cell surface As NBS- modified ECP inhibited the growth of A431 cells less than (—4) ECP and the ICs, could not be determined (T Maeda,

D L Newton, S M Rybak, unpublished results), the affinity for heparin must also be responsible for the growth- inhibitory effect

DISCUSSION

This is the first report to demonstrate that ECP has a growth-inhibitory effect that is cytostatic and dependent on cell type The growth of four of the seven human cell lines

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 pm ECP (D, E, F) or RNase A (G, H, ID) 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 magnifications are x 10

Fig 5 Growth-inhibitory effect 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 um 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%

ECP RNaseA

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 um each RITC-labeled ECP and RNase A was added to the culture medium, and the cells were fixed and detected by fluorescent 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 field was assigned in the same column at each time The scale bar is equivalent to 50 um

A

=

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—

©

®©

o

°

2

‹

-0.20

30 40 50 60 70 Retention time (min)

Fig 7 Heparin affinity column chromatography of ECP (A) and schematic diagram of ECP depicted with RasMol v2.6 according to the PDB entry IDVT (B) (A) ECP modified with NBS (a) (—4) ECP (b) or ECP (c) was applied to a heparin column and eluted with a linear gradient of NaCl The retention time of each peak of profiles is 43 min (0.64 m NaCl) for NBS-modified ECP, 48 min (0.60 Mm NaCl) for (—4) ECP and 53 min (0.56 Mm NaCb for ECP RNase A passed through this affinity column under the same conditions (B) The backbone of peptide bonds is drawn in gray The secondary-structure elements are helices and arrows for « helices and B strands, respectively Two tryptophan residues, W10 and W385, and the other amino-acid residues in the catalytic subsites are in black with their side chains H64 is located in the PO subsite, H15, K38 and H128 in the P1 subsite, and W1O in the P2 subsite

was inhibited, whereas the rodent cell lines were relatively

resistant The resistance of the rodent cell lines to the

growth-inhibitory effects of human ECP may be due to the

evolutionary divergence of ECP, which resulted in signifi-

cantly low homology of eosinophil-derived RNase between

species [46,47] In this study, ECP suppressed the growth of

K562, HL-60, A431 and MDA-MB-453 cell lines in an

ICs9 range of 1-4 um Although the primary structure of

human 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 extension

of 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 Cancer Institute, National Institutes of Health, Frederick, MD, USA) On the contrary, the N-terminal extension of ECP appears to impair the inhibitory effect of ECP on some cell lines

ECP is unique among RNase A enzymes because of its

high arginine content Therefore, the nonspecific effect of

growth 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 that

of ECP It did not have any growth-inhibitory effect on

K562 and A431 cells (Fig 2) Although we could not obtain

polyarginine, we assessed the effect of the third helix of the

Drosophila antennapedia homeodomain [48] and the basic

region of HIV-Tat protein [49], both of which are rich in

basic amino acids and known to enhance cellular uptake

Neither of these peptides showed any growth-inhibitory

effect similar to ECP (data not shown) Therefore, the

cationic charge could not replicate the effect of ECP

Recently, it has been shown that the amphipathic helix

structure of the basic region of Tat protein may be

important for the uptake of the protein into cells [50] This

mechanism may not apply to ECP as ECP is an arginine-

rich protein but does not have the cluster of arginine

residues like Tat protein On the other hand, highly

cationized RNase molecules are cytotoxic but are not

cytostatic like ECP [51]

The cytostatic growth-inhibitory effect of ECP was not

sufficient to induce cell death, as the initial number of viable

K562 cells did not significantly decrease in the presence of

ECP Cell cycle analysis showed that ECP decreased K 562

cells in the G;/Go phase without having an effect on the G>/

M phase The number of dying cells increased slightly but

this was not significant The growth-inhibitory effect of ECP

was reversible as A431 cells treated with ECP resumed

normal growth when ECP was removed from the medium

In addition, ECP affected the size of colonies of A431 cells

but not colony number Furthermore, we found no effect of

ECP on the frequency of apoptosis as assessed by DNA

ladder formation and phosphatidylserine externalization

(data not shown)

RITC-labeled ECP appeared to accumulate in the cyto-

plasm after 24 h However, whether endocytosis of ECP

ocurred is still unclear It may be in vesicles or endosomes As

the cellular outlines were not enhanced during the course of

the incubation, we concluded that specific accumulation of

ECP on the cell surface was not occurring The most probable

explanation for this observation is the presence of cell sur-

face receptors for ECP We could not show the presence of a

specific high-affinity binding site for ECP on cells by com-

petition assay This is probably because the affinity of ECP

for binding to extracellular matrix proteins such as heparan

sulfate is extremely low and rapidly dissociates However, it

is very interesting to note that ECP can bind to heparin and

that tryptophan residues contribute to this affinity As tryp-

tophan residues are also responsible for the affinity for

galactose and lactose residues of some lectins [52], it is feasible

that this is also the case for ECP The tryptophan residues

are Trpl0 and/or Trp35 in ECP Interestingly, Trp10 is

located at the P2 subsite of the catalytic domain of RNase

and controls the weak RNase activity of the protein [6]

Trp35 is a unique amino-acid residue at a position in

Loop-3 of the RNases As both tryptophan residues are

located on the same side of the molecule, it is difficult to

ascertain which residue is more critical for the the binding of

ECP to heparin or other carbohydrates

In conclusion, ECP has a cell-type-specific cytostatic

growth-inhibitory effect It is possible that this activity is due

to the presence of the tryptophan residues We are now producing mutant ECP proteins to assess this aspect Further evaluation of the ECP molecule including the generation of a mutant protein will enable us to test the effects of ECP on inflammatory diseases, in which uncon- trolled cell growth could contribute to a delay in wound healing In addition, strong local inflammatory responses that are specific to cytokines such as interleukin-4, are capable in some cases of mediating regression of tumors [53,54] Inflammatory infiltrates comprised of eosinophils may also play an important role at the primary site of tumor regression by releasing ECP as part of the cascade of inducing a tumor-specific T-cell response Optimization of the biological activity of the ECP protein to target the cell surface could bring ICs9 values down to practical levels so that it might be used as an anti-cancer reagent in the same way as others [27,55—57]

ACKNOWLEDGEMENTS

We thank Drs S Rybak and D Newton for assaying the effects of ECP and (—4) ECP on KS Y-1 cells, Professor K Oguma for providing

S aureus cells, Professor M Hikida for help with flow-cytometric analysis, and Drs R Sasada, S Ishikami and J Futami for helpful discussions and suggestions throughout this work This work was partly supported by a grant-in-aid from the Ministry of Education, Science, Culture and Sports of Japan R de LI was supported by the Spanish Ministerio de Educacion y Cultura (grant SAF 98-0086 and 2FD97- 0872) and by Generalitat de Catalunya (grant SGR97-240)

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