Eur. J. Biochem. 269, 307–316 (2002) (cid:211) FEBS 2002
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
isothiocyanate-labeled ECP started to bind to A431 cells after 0.5 h and accumulated for up to 24 h, indicating that specific a(cid:129)nity for the cell surface may be important. The a(cid:129)nity 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 e(cid:128)ect 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 e(cid:128)ect; eosinophil cationic protein (ECP); growth inhibition.
Eosinophil cationic protein (ECP), one of the major components of basic granules of eosinophils, is cytotoxic to tracheal epithelium. However, the extent of this e(cid:128)ect on other cell types has not been evaluated in vitro. In this study, we evaluated the e(cid:128)ect 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 IC50 values on A431 cells, MDA-MB-453 cells, HL-60 cells and K562 cells were esti- mated to be (cid:25) 1–5 lM. ECP significantly suppressed the size of colonies of A431 cells, and decreased K562 cells in G1/G0 phase. However, there was little evidence that ECP killed cells in either cell line. These e(cid:128)ects of ECP were not enhanced by extending its N-terminus. Rhodamine B
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.
M A T E R I A L S A N D M E T H O D S
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 in 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 1 is (cid:25) 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 (cid:25) 100–2000-fold less active than EDN depending on the type of substrate [8,9].
Cell cultures
ECP is bactericidal [10], helminthotoxic [11–17], elicits the Gordon phenomenon when injected intrathecally into
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
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: senom@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)
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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
X-100 and 0.15 mg(cid:1)mL(cid:255)1 RNase A for 15 min at room temperature. The cells were then fixed in 70% ethanol overnight at 4 (cid:176)C and this was followed by further treatment with RNase A (0.1 mg(cid:1)mL(cid:255)1) in NaCl/Pi for 10 min at 37 (cid:176)C. The DNA of the fixed cells was stained with propidium iodide (50 mg(cid:1)mL(cid:255)1) 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 lM 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
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 ((cid:255)4) ECP. Purified ECP and ((cid:255)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 ((cid:255)4) ECP, which was processed off.
MTT assay for cell growth
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 1 lM for 0.5–24 h, then washed with NaCl/Pi, and observed under a fluorescent microscope (BX40; Olympus). Hoechst 33342 dissolved in NaCl/Pi to 2 lM (Molecular Probes) was used for nuclear staining.
Oxidation of tryptophan residues
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 lM). 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(cid:1)mL(cid:255)1 in NaCl/Pi) was added, and cell growth was monitored by measuring A570.
Counting of viable cells
Two tryptophan residues were modified by oxidation with N-bromosuccinimide (NBS; Sigma) as previously described [41]. Briefly, 1.37 mg(cid:1)mL(cid:255)1 NBS dissolved in 50 mM sodium acetate, pH 4.5, was gradually added to a solution of ECP (1.6 mg(cid:1)mL(cid:255)1) in the same buffer. Oxidation was monitored by measuring the decrease in A280 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.
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 ((cid:255)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.
Heparin-affinity column chromatography
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 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) equipped with a charge-coupled device video camera.
ECP with or without modification was applied to a heparin affinity column (Heparin-Cellulofine; 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 per 60 min) at a flow rate of 0.6 mL(cid:1)min(cid:255)1. The A273 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.
Cell cycle analysis
R E S U L T S
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
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 lM ECP or bovine RNase A (Sigma). After 3 days of treatment, cells were harvested, washed with NaCl/Pi, treated with NaCl/Pi containing 0.25% Triton
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against S. aureus whereas RNase A did not show any activity (Fig. 1C). Therefore, both forms of recombinant ECPs were biologically active.
Effect of ECPs on various cell lines
The growth-modulatory effects of ECP and ((cid:255)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 IC50 (concentration that causes 50% inhibition) of 1.1 lM. A431, MDA-MB-453 and HC-11 cells were also sensitive to ECP with IC50 values of (cid:25) 4–6 lM. The ((cid:255)4) ECP protein also inhibited the growth
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 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 ((cid:255)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 ((cid:255)4) ECP showed no difference in RNase activity (Fig. 1B). Both forms of ECP exhibited bactericidal activity
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 purified from eosinophils. Recombinant ECP was prepared as the mature form starting from Arg28 as indicated by the arrow at the top, and ((cid:255)4) ECP was 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 ((cid:255)4) ECP (j) were eval- uated. Bovine RNase A (s) is a control.
Table 1. E(cid:128)ect of ECP and ((cid:255)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 lM ECP or ((cid:255)4) ECP; NT, not tested; NA, not applicable.
((cid:255)4) ECP ECP
IC50 (lM) SC (%) IC50 (lM) SC (%) Cell line Origin
Human K562 HL-60 A431 MDA-MB-453 TE-8 T-47D HT-29 Chronic myelogenous leukemia Acute promyelocytic leukemia Epidermoid carcinoma Breast carcinoma (mammary gland) Squamous carcinoma Ductal carcinoma (mammary gland) Colon adenocarcinoma 1.1 1.1 4.0 4.0 NA NA NA 1.7 (cid:139) 0.3 2.1 (cid:139) 0.5 38.7 (cid:139) 7.6 31.3 (cid:139) 4.7 68.5 (cid:139) 2.8 90.3 (cid:139) 3.9 75.3 (cid:139) 4.7 2.0 2.0 6.0 NT NT NT NA 3.0 (cid:139) 1.3 5.4 (cid:139) 2.4 49.3 (cid:139) 9.7 NT NT NT 80.5 (cid:139) 2.8 Mouse
B16-BL6 LL/2 3T3-SV40 SV-T2 HC-11 Metastatic melanoma Lewis lung carcinoma SV-40-transformed Swiss 3T3 cells SV-40-transformed Balb/c 3T3 cells Normal mammary gland epithelial cells NA NA NA NA 6.0 73.5 + 9.0 93.5 (cid:139) 5.5 57.6 (cid:139) 9.8 90.3 (cid:139) 8.7 42.8 (cid:139) 5.2 NT NA NA NA 6.0 NT 100.6 (cid:139) 4.5 58.9 (cid:139) 8.5 95.2 (cid:139) 8.2 38.3 + 4.4 Rat A10 Normal aortic smooth muscle cells NA 84.5 + 2.3 NT NT
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of these cell lines. However ((cid:255)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 IC50 values could not be accurately calculated but were more than 10 lM.
Effect of ECP on A431 and K562 cells
After 7 days of incubation, ECP and ((cid:255)4) ECP appeared to inhibit the aggregation of K562 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 ((cid:255)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
At 5 lM ECP, the growth of K562 cells was completely suppressed whereas growth inhibition of A431 cells was (cid:25) 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(L-lysine) (average molecular mass (cid:136) 2900). Neither RNase A nor poly (L-lysine) had any effect on the growth of these cells (Fig. 2).
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), ((cid:255)4) ECP (j) and ECP (d) 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, 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 each RNase A (B), ECP (C) and ((cid:255)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 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 regular medium without ECP (H). (G) and (H) show the same field of the same dish. Original magnifications of the plates are · 10.
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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 (cid:139) 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. 5A). However, ECP produced a significant decrease in 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 (cid:176)C for various times (Fig. 6). From 0.5 to 3 h, 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 lM 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 lM. 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 M NaCl, and ((cid:255)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 ((cid:255)4) ECP and the IC50 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.
Fig. 3. Growth-inhibitory e(cid:128)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 ((cid:255)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 lM 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 G1/G0 and G2/M phase, respectively.
D I S C U S S I O N
supplemented by the medium change, this effect was assessed in the first 3 days of treatment by monitoring the cell number (Fig. 3A). ((cid:255)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 in the population of cells in the G1/G0 phase of the cell cycle, and a small increase in 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 G2/M phase of the cell cycle was not altered, and the total cell number was unaffected. A small number of K562 cells in the G1/G0 phase of the cell cycle that had been treated with ECP or ((cid:255)4) ECP appeared to be dead.
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
The effect of ECP in the early period without medium change was monitored on A431 cells (Fig. 4). Up to 4 days
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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 magnifications are · 10.
Fig. 5. Growth-inhibitory e(cid:128)ect of ECP on A431 cells. A431 cells were seeded at 5000 cells per 35-mm dish, and 2500 (cid:139) 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%.
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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 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 lm.
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- line (personal communi- inhibitory effects on this cell 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.
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 IC50 range of 1–4 lM. Although the primary structure of human ECP shows the closest identity (67%) with human
Fig. 7. Heparin a(cid:129)nity column chromatography of ECP (A) and schematic diagram of ECP depicted with RasMol v2.6 according to the PDB entry 1DVT (B). (A) ECP modified with NBS (a) ((cid:255)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 M NaCl) for ((cid:255)4) ECP and 53 min (0.56 M NaCl) for ECP. RNase A passed through this a(cid:129)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 a helices and b strands, respectively. Two tryptophan residues, W10 and W35, and the other amino-acid residues in the catalytic subsites are in black with their side chains. H64 is located in the P0 subsite, H15, K38 and H128 in the P1 subsite, and W10 in the P2 subsite.
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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 IC50 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].
A C K N O W L E D G E M E N T S
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].
We thank Drs S. Rybak and D. Newton for assaying the e(cid:128)ects of ECP and ((cid:255)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 Ll. was supported by the Spanish Ministerio de Educacio´ 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
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 K562 cells in the G1/G0 phase without having an effect on the G2/ 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).
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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 Trp10 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.
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