Molecular Biology, Vol. 39, No. 3, 2005, pp. 404–409. Translated from Molekulyarnaya Biologiya, Vol. 39, No. 3, 2005, pp. 457–463. Original Russian Text Copyright © 2005 by Zelenikhin, Kolpakov, Cherepnev, Ilinskaya.
CELL MOLECULAR BIOLOGY UDC 577.1;581.17
Induction of Apoptosis in Tumor Cells by Binase P. V. Zelenikhin1, A. I. Kolpakov1, G. V. Cherepnev2, and O. I. Ilinskaya1,3 1 Kazan
State University, Kazan, 420008 Russia e-mail:
[email protected] 2 Institute of Medical Immunology of Charite, Berlin, 10117 Germany 3 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia Received December 27, 2004
Abstract—The possibility of inducing apoptosis in K562 myelogenic erythroleukemia cells, A549 lung carcinoma cells, and normal human lymphocytes was studied for Bacillus intermedius RNase (binase) and its mutants Lys26Ala and His101Glu with impaired catalytic activity. Selective induction of apoptosis in leukemic blood cells by binase was demonstrated for the first time. Binase did not exert an antiproliferative or proapoptotic effect on peripheral blood lymphocytes of healthy donors. Low-molecular-weight (less than 50 kb in size) oligonucleosomal DNA fragments, which are early markers of apoptosis, were observed in human solid-tumor cells treated with binase. Studies with the binase mutants showed that a decrease in catalytic activity to 2.5% of the level characteristic of the wild-type enzyme deprives binase of its proapoptotic effect. The selective proapoptotic effect of binase on malignant cells provides evidence that bacterial RNases are promising for designing alternative antitumor drugs. Key words: cytotoxic RNases, binase, Lys26Ala and His101Glu binase mutants, K562 myelogenic erythroleukemia cells, A549 lung carcinoma cells, lymphocytes, apoptosis
INTRODUCTION The increasing incidence of cancer requires a search for new drugs and development of new approaches to antitumor therapy. Interest in ribonucleases (RNases) of various origins has recently quickened, because some of these enzymes have a considerable antitumor activity. RNase of frog Rana pipiens oocytes is highly cytotoxic and induces apoptosis in many tumor cell lines. Onconase, a commercial preparation of this RNase, is at stage III of clinical trials as a new means of therapy for lung mesothelioma [1]. Bovine seminal RNase (BS-RNase), acting as a dimer tolerant of the mammalian cytosolic RNase inhibitor, exerts an antitumor effect toward thyroid cancer and neuroblastoma cells [2, 3]. A toxic effect on cancer cells is characteristic of fungal RNase, in particular, α-sarcin [4]. There is evidence that RNase Sa3 secreted by Streptomyces aureofaciens is cytotoxic for myeloleukemia cells [5]. Bacillus intermedius RNase (binase) suppresses the growth of Erlich ascite and NKLy leukemia cells in experimental animals [6]. It should be noted that bacterial RNases have several advantages over mammalian RNases: bacterial enzymes are not inactivated by the cytosolic inhibitor and are less expensive to isolate. The capability of selectively inducing apoptosis in tumor cells is of primary importance for RNase to be
employed in cancer therapy. The selectivity of action is mostly due to the cationic character of RNases, since cancer cells carry far more negatively charged phospholipids, glycolipids, and glycoproteins on their surface than normal cells [7]. The electrostatic interaction of RNases with these compounds makes their internalization more likely. Indeed, the vast majority of RNases that exert the cytotoxic effect and induce apoptosis in tumor cells are cationic proteins [8]. For instance, onconase (pI 10.3) activates the mitochondrial pathway of caspase-dependent apoptosis in human leukemia cells [9], and BS-RNase (pI 10.4) induces apoptosis in thyroid cancer cells [10]. Binase is also a cationic protein (pI 9.5) and is toxic predominantly for cells transformed with the ras oncogene [11], which is expressed in many human and animal tumors [12]. However, induction of apoptosis by this RNase has not yet been demonstrated. Apoptosis is considered to be an early stage of toxic cell damage associated with an impaired capability of repairing DNA lesions and changes in gene expression [13]. In view of this, it is important to note that RNase interactions with a substrate without its catalytic cleavage can also change gene expression, as observed for the binding of bacterial RNase III with double-stranded RNA [14]. Thus, the proapoptotic effect of RNases is associated both with RNA hydrolysis and with RNA noncatalytic binding, which is
0026-8933/05/3903-0404 © 2005 Pleiades Publishing, Inc.
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Catalytic activity of binase and its Lys26Ala and His101Glu mutants and changes in morphological and physiological characteristics of A549 cells upon culturing in the presence of RNases for 72 h Catalytic activity RNase
RNA, untis/mg (%)*
poly(I), M–1 s–1 (%)**
Binase Lys26Ala His101Glu Without RNase
14 × 106 (100) 4 × 106 (28) 0.3 × 106 (2.4)
7.2 (100) 2.4 (33) 0.15 (2.1)
Cell number, 103 per dish
Dehydrogenase activity (MTT test), %
Apoptotic nuclei, %***
Anoikis
560 ± 5 990 ± 10 1240 ± 8 1260 ± 10
40 ± 4 75 ± 8 90 ± 7 100
15 ± 5 7±2 5±2 4±1
+ – – –
* According to [15] ** kcat /KM according to [16]. *** Total number of cells/nuclei in a dish was taken as 100%.
easy for cationic molecules owing to the anionic character of nucleic acids. The objectives of this work were to study the apoptosis-inducing activity of binase and to estimate the contribution of its catalytic activity to induction of apoptosis. To evaluate the selectivity of binase action, its proapoptotic effect was compared for human solid tumor cells and malignant and normal blood cells. EXPERIMENTAL Enzymes. We used the wild-type B. intermedius 7P binase (12.3 kDa, pI 9.5) and its mutant variants Val26Ala and His101Glu (constructed and kindly provided by G.I. Yakovlev, Engelhardt Institute of Molecular Biology) [15]. The catalytic activities of these enzymes with synthetic substrates [15] and highmolecular-weight yeast RNA [16] were characterized earlier (table). Cell cultures. The proapoptotic effects of binase and its mutants were assayed with K562 myelogenic erythroleukemia cells (Institute of Medical Immunology of Charite); A549 human lung carcinoma cells (American Type Culture Collection, United States); and peripheral mononuclear leukocytes, which were isolated by centrifuging blood of healthy donors through Ficoll–verographin (Pharmacia, Sweden) according to the standard protocol [17]. K562 and mononuclear cells were cultured in RPMI 1640; A549 cells were cultured in the HamF12K medium, both media supplemented with 10% bovine fetal serum (Sigma, United States), 2 mM glutamine, 100 units/ml penicillin, and 100 units/ml streptomycin. Cell cultures were grown in an atmosphere containing 5% ëé2 at 37°C. Apoptotic changes in blood cells were studied with a FACSCalibur flow cytometer (United States). Cells were cultured for 48 h in the presence of RNases and double-stained with MC540 (Sigma, United States), which binds to phosphatidylserine residues on MOLECULAR BIOLOGY
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the outer membrane surface of apoptotic cells [18], and 7-aminoactinomycin D (7-AAD, Sigma, United States), which interacts with degrading DNA at sites between guanine and cytosine [19]. Phosphatidylserine residues, which are normally restricted to the inner surface of the plasma membrane, are transferred onto the outer surface during apoptosis and become accessible to MC540. Staining with MC540 reports apoptotic (MC540-positive) and normal (MC540-negative) cells. At the late stage of apoptosis, the integrity of cell membranes is impaired and, consequently, 7-AAD penetrates through membranes into the cell nucleus and binds to DNA fragments. Thus, double staining with MC540 and 7-AAD reveals three cell populations: (1) MC540-negative 7-AAD-negative normal cells, (2) MC540-positive 7-AAD-negative early apoptotic cells, and (3) MC540-positive 7-AADpositive late apoptotic cells (Fig. 1). Apoptotic changes in solid tumor (A549) cells were detected by fluorescence microscopy; DNA fragmentation was assayed by pulsed-field electrophoresis. To visualize cell nuclei, cells were fixed with methanol and stained with Hoechst 33358. A549 cells were labeled with [3H]thymidine during exponential growth and incubated with RNase for 24, 48, or 72 h. Electrophoresis was carried out according to the standard protocol in a BioRad CHEF-DRIII system [20]. To study the distribution of the label through DNA fractions, gel was divided into regions and the radioactivity of each region was measured in a Minaxi TriCarb 4000 counter (Packard). As markers, we used restriction fragments of Saccharomyces cerevisiae (0.24–2.2 Mb) and phage λ (145.5, 97, and 48.5 kb) DNAs. Gels were stained with 0.5 µg/ml ethidium bromide and photographed in UV light. Mitochondrial dehydrogenase activity, which decreases together with the metabolic activity of the cell, was assayed colorimetrically with 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) [21]. In parallel, cells were counted in a chamber under a microscope.
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ture, and 83% in the presence of camptothecin. By contrast, the portion of early apoptotic cells increased with increasing binase concentration and, at the maximal concentration, reached 31% versus 5% in the control and 15% in the presence of camptothecin. Likewise, the proportion of late apoptotic cells increased from 5 to 13% as binase concentration was increased from 30 to 750 µg/ml (Fig. 2). This proportion was 1% in the absence of the enzyme and 2% in the presence of camptothecin. Thus, binase used in micromolar concentrations showed a considerably higher proapoptotic activity toward K562 cells than 50 mM camptothecin.
(‡)
104 IV
III
I
II
7-AAD
103
102
101
100 100
101
102 MC540
103
104
(b)
Normal cell
Apoptotic cell
Fig. 1. Cytometric distribution of K562 cells by populations revealed by double staining with MC540 and 7-AAD: (a) I, normal cells; II, early apoptotic cells; III, late apoptotic cells; and IV, necrotic cells. (b) The populations of normal and late apoptotic cells were checked morphologically.
As a positive control, 50 mM camptothecin (Sigma) was used in experiments on induction of apoptosis [22]. Statistical analysis of the results was performed using the Statistica 6.0 program. The significance of differences was evaluated by the Mann–Whitney nonparametric test; the difference was considered significant at p ≤ 0.05. RESULTS AND DISCUSSION Binase showed a dose-dependent apoptosis-inducing effect and suppressed proliferation of K562 cells at 30–750 µg/ml (Fig. 2). The toxic effect of binase was evident from a decrease in the total cell number. When cells were incubated for 48 h in the presence of 30, 150, or 750 µg/ml binase, their count decreased by 24, 37, and 42%, respectively. Camptothecin also exerted a toxic effect and decreased the cell count by 47% as compared with control samples. Binase reduced not only the total cell number, but also the portion of viable nonapoptotic cells. Normal cells accounted for 56% of the K562 cell population in the presence of 750 µg/ml binase, 94% in a control cul-
The concentration dependence of the effect of the Lys26Ala mutant, which preserved about 30% of the initial RNase activity (table), did not differ significantly from that of the wild-type enzyme (Fig. 2). Thus, the catalytic activity of this mutant sufficed to induce apoptosis. Although many cationic peptides, such as wellknown mammalian factors of antibacterial defense, exert a selective proapoptotic effect on tumor cells [23], the cationic character is insufficient for induction of apoptosis. We checked this assumption with K562 cells and lysozyme, an antibacterial defense protein that is similar to binase in molecular weight (14 kDa) and bears a high positive charge (pI 11). In the concentration range examined (6–750 µg/ml), lysozyme neither induced apoptosis nor inhibited cell proliferation (Fig. 2). Our results make it possible to conclude that the proapoptotic and antiproliferative effects of binase depend on its specific molecular determinants, in particular, on its RNase activity. This assumption agrees with other data on the mechanisms of apoptosis: cleavage of various RNAs always accompanies apoptosis and usually precedes DNA degradation [24, 25]. The positive charge of an RNase molecule plays an accessory role, facilitating its interaction with negatively charged groups on the cell surface; internalization; and, possibly, the binding with accessible RNAs within the cell. We compared the toxic effect on A549 lung carcinoma cells for binase and its mutants. Both wild-type binase and the Lys26Ala mutant caused morphological changes in A549 cells, but the mutant showed lower toxicity and a lower proapoptotic effect (table). The mutant with the substitution His101Glu in the catalytic center was nontoxic: the portion of apoptotic cells in an A549 culture treated with this mutant was no more than that in a nontreated culture. Generation of lowmolecular-weight (less than 50 kb in size) DNA fragments was observed only in A549 cells treated with wild-type binase (500 µg/ml) for 72 h (Fig. 3a). Quantitation of the label incorporated into DNA showed that the peak of radioactivity corresponded to fragments of 20–150 kb, which accounted for no more than 6% of the total DNA. Cell viability in cultures MOLECULAR BIOLOGY
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1200 1%
1%
Total count, 103 cells/ml
1000
5%
1%
5%
LA EA N
1% 5%
8% 5%
800
5%
9%
9% 4%
600
1% 8%
5% 13% 18%
20%
12% 2%
3%
400
15% 31%
3%
35%
200 94%
0
94%
94%
K K B 0 h 48 h 6
94%
91%
86%
M 6
L 6
B 30
86%
76%
77%
56%
53%
91%
83%
M B M B M L 30 150 150 750 750 750 µg/ml
C
Fig. 2. Distribution of K562 cells by the late apoptotic (LA), early apoptotic (EA) and normal (N) populations. Cells were examined before and after 48-h culturing in the absence (K, control) or presence of wild-type binase (B), its Lys26Ala mutant (M), lysozyme (L), or camptothecin (C).
treated with binase decreased by 60%, to 40% of the viability characteristic of nontreated control cultures (table). Morphologically, cells treated with binase only slightly differed from normal cells; late apoptotic nuclei were observed in about 15% of cells (Fig. 3b). Apoptosis is known to involve nuclear picnosis, which results in generation of oligonucleosomal DNA fragments producing an about 18-kb ladder upon agarose gel electrophoresis. Before such fragments are generated, fragments of 20–300 kb became detectable as markers of early apoptosis [26]. It is this state that was observed for cells cultured in the presence of binase for 72 h. Thus, binase acts as a proapoptotic agent on cells of solid tumors, but its effect is weaker than that on erythroleukemia cells. Epithelial cells are known to undergo anoikis, a special form of apoptosis associated with cell detachment from the extracellular matrix [27]. We observed that A549 cells tended to separate from the monolayer in cultures treated with binase, testifying again to induction of apoptotic changes. The binase mutants with lower catalytic activities neither exerted a toxic effect nor induced apoptosis in A549 cells. Thus, a higher catalytic activity of binase is required to damage cells of solid tumors than erythroleukemia cells. To study the possibility of the cytotoxic effect of binase on normal human cells, we used lymphocytes isolated from the peripheral blood of healthy donors. At the concentration range examined (6–750 µg/ml), binase did not induce apoptosis in these cells (Fig. 4). Likewise, no change in physiological parameters of normal lymphocytes was observed upon treatment with the Lys26Ala mutant. This finding suggests a selective effect of binase on tumor cells. The selectivity of binase is first and foremost due to its cationic character, which ensures high affinity for anionic groups exposed on the surface of tumor cells. Another MOLECULAR BIOLOGY
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available means to improve the selectivity of RNases is to link them to specific vectors, such as monoclonal antibodies, which interact with certain surface components of tumor cells [28]. Cell treatment with the envelope protein of a hemagglutinating virus and protamine sulfate increases cell permeability and allows the intake of bacterial RNase T1, which is nontoxic for normal cells because of a lack of internalization. However, the enzyme induces apoptosis in normal as well as in tumor cells treated in such a way [29]. Apo-
kb
565 450 365 285 220
Sc
(a) Bi K λ
(b) kb
Nucleus of normal cell 145.5 97 48.5 Nucleus of late apoptotic cell
Fig. 3. (a) DNA fragmentation induced by binase in A549 cells. Lanes: Sc, marker fragments of S. cerevisiae DNA; Bi, DNA of cells treated with 0.5 mg/ml binase for 72 h; λ, marker fragments of phage λ DNA; and K, DNA of nontreated control cells grown for 72 h. (b) Nuclei of A549 cells grown for 72 h in the presence of 0.5 mg/ml binase.
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600 1% Total count, 103 cells/ml
500
1%
1% 10%
1% 10%
7%
30%
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REFERENCES
300 200 89%
92%
89%
69%
K 48 h
B 6
B 750
C
LA EA N
100 0
no. LST.CLG.979534), the program Russian Universities—Basic Research (project no. ur.11.01.004), and the Foundation of the Academy of Sciences of Tatarstan (project no. 03-3.10-292).
µg/ml
Fig. 4. Distribution of lymphocytes through the late apoptotic (LA), early apoptotic (EA) and normal (N) populations. Cells were examined after 48-h culturing in the absence (K, control) or presence of wild-type binase (B) or camptothecin (C).
ptosis is associated with the activation of cell RNases. For instance, the interferon-inducible apoptosis pathway leads to activation of 2',5'-oligoadenylate-dependent RNase L [30], degradation of rRNAs induces caspase-dependent apoptosis [31], and RNase activity of α-RNP particles in K562 cells increases in response to the apoptosis inductors doxorubicin and diethyl maleate [32]. It is thought that damaging RNA to induce apoptosis is a promising approach to cancer therapy [33]. Studies have shown that inactivation of binase reduces its cytotoxicity [34] and that the in vivo antitumor effect does not depend on the catalytic activity in animals [6]. The latter finding is probably explained by the fact that intratumor injection allows a higher penetration of RNase into cells; consequently, it is more likely that the enzyme changes gene expression and induces apoptosis by noncatalytically interacting with RNA or that the inactivated enzyme still has a residual RNase activity within the cell. Thus, we showed that binase selectively induces apoptosis in tumor cells. The catalytic activity of binase makes the major contribution to its proapoptotic effect, testifying that cleavage of accessible RNA is essential not only for the toxic but also for the proapoptotic effect. ACKNOWLEDGMENTS We are grateful to G.I. Yakovlev for the enzymes and to F. Kern for a position in his laboratory. This work was supported by the Foundation of the Berlin Chamber of Deputies, the program “Molecular and Cell Biology” of the Russian Academy of Sciences, the Russian Foundation for Basic Research (project no. 04-04-49385), the NATO Foundation (grant
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