Báo cáo khóa học: Noncytotoxic ribonuclease, RNase T1, induces tumor cell death via hemagglutinating virus of Japan envelope vector

doi:10.1111/j.1432-1033.2004.04293.x

Eur. J. Biochem. 271, 3567–3572 (2004) (cid:1) FEBS 2004

Noncytotoxic ribonuclease, RNase T1, induces tumor cell death via hemagglutinating virus of Japan envelope vector

Shunji Yuki, Yoshitaka Kondo, Fuminori Kato, Masanari Kato and Norifusa Matsuo

Central Research Institute, Ishihara Sangyo Kaisha Ltd, Kusatsu, Shiga, Japan

cancer chemotherapeutic agent. Furthermore, internalized RNase T1 induced apoptotic cell death programs. Because its cytotoxicity is unfortunately not specific to tumor cells, it cannot at present be developed as an anticancer drug. However, we believe that RNase T1 incorporated in HVJ envelope vector will be a unique anticancer drug if HVJ envelope vector can be targeted to tumor cells.

Keywords: apoptosis; HVJ envelope vector; protein trans- fection; RNase T1; tumor cell death.

Several ribonucleases, including onconase and a-sarcin, are known to be toxic to tumor cells. On the other hand, although its structure is related to that of a-sarcin, RNase T1 is noncytotoxic because of its inability to internalize into tumor cells. In this study, we internalized RNase T1 into human tumor cells via a novel gene transfer reagent, hem- agglutinating virus of Japan (HVJ) envelope vector, which resulted in cell death. This cytotoxicity was drastically increased by pretreatment of HVJ envelope vector with protamine sulfate, and was stronger than that of onconase, which is in phase III human clinical trials as a nonmutagenic

proteins to enhance the interactions between RNases and the plasma membrane [7]. The cytotoxicity of such targeted RNases has been enhanced by several orders of magnitude. For instance, onconase cytotoxicity for a human B-cell tumor cell line was increased 10 000-fold with coupling to an LL2 antibody, specifically binding the CD22 antigen [8]. Alternatively, some researchers have used carriers such as liposomes to enhance the internalization of RNases. Iordonov et al. reported that HeLa cells were efficiently killed by Lipofectin-mediated delivery of onconase, though they did not aim at its clinical use [9].

Ribonucleases are best known for their ability to cleave RNA. However, some of these proteins have been shown to be much more than digestive enzymes. For example, two groups of these enzymes have antitumor activities [1–4]. One is the family of fungal ribotoxins that specifically cleave one phosphodiester bond of the larger rRNA at a highly conserved region, inhibit protein synthesis and eventually cause cell death [3,4], while the other is composed of certain members of the bovine pancreatic RNase A superfamily. Onconase, a homolog of RNase A from the Northern leopard frog, Rana pipiens, is selectively toxic to cancer cells both in vitro and in vivo, and is now being evaluated as a cancer chemotherapeutic agent in phase III clinical trials for mesothelioma [1].

In order to kill cells, internalized RNase must reach the cytosol, where RNA exists. Bovine seminal RNase, a dimeric RNase selectively cytotoxic for malignant cells, is endocytosed and internalized in endosomes in both normal and malignant cells, but reaches the cytosol only in malignant cells [10]. RNase cytotoxicity therefore requires not only its efficient internalization but its correct localiza- tion.

The first step in RNase cytotoxicity is the internalization of the protein across the phospholipid bilayer barrier. a-Sarcin, the best characterized member of the family of fungal ribotoxins, directly interacts with the phosphlipids of the cell membrane and translocates into the cytosol by endocytosis [5]. Onconase binds to specific but unknown receptors on the plasma membrane of the target cell and enters it by endocytosis [6]. They undergo internalization intrinsically, but their translocation across the plasma membrane is the rate-limiting step of the cytotoxic effect [6]. Therefore, many research groups have created fusion

Correspondence to S. Yuki, Central Research Institute, Ishihara Sangyo Kaisha Ltd, 2-3-1 Nishi-Shibukawa, Kusatsu, Shiga, 525-0025, Japan. Fax: +77 562 9752, Tel.: +77 562 8999, E-mail: s-yuki@iskweb.co.jp Abbreviations: AU, assay units; CML, chronic myelogenous leukae- mia; FBS, fetal bovine serum; HVJ, hemagglutinating virus of Japan; 1-methoxy PMS, 1-methoxy-5-methylphenazinum methylsulfate; PS, protamine sulfate; WST-1, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5- (2,4-disulphophenyl)-2H-tetrazolium monosodium salt. (Received 30 March 2004, revised 30 June 2004, accepted 19 July 2004)

Recently, Kaneda and colleagues developed a hemagglu- tinating virus of Japan (HVJ; Sendai virus) envelope- mediated gene transfer system [11]. Because in this system inactivated HVJ envelope (HVJ envelope vector) containing plasmid DNA is efficiently fused with the plasma membrane of target cells, the plasmid DNA is easily and directly introduced into the cytosol without degradation by endo- somes and lysosomes. Therefore, we expected that the HVJ envelope vector trapped RNase could become an effective antitumor agent.

RNase T1 from the fungus Aspergillus oryzae is a small acidic protein structurally related to a-sarcin but nontoxic to tumor cells [12]. The tapetum is selectively destroyed in transgenic plants that express the RNase T1 gene specific- ally in anther tapetum [13]. These findings suggest that RNase T1 is noncytotoxic but that its efficient introduction into target cells could cause their death. We therefore considered RNase T1 a good model for evaluating the effect

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of HVJ envelope vector on RNase cytotoxicity, prepared RNase T1-containing HVJ envelope vector and investi- gated its cytotoxicity for tumor cell lines.

Experimental procedures

Materials

Cytotoxicity assay K-562 (1 · 104 cells per well) or SAS (4 · 103 cells per well) cells were seeded in a 96 well plate. Following 24 h incubation in 200 lLÆwell)1 of culture medium, RNase T1 alone (10 lLÆwell)1) or RNase T1 incorporated in HVJ envelope vector (2 lLÆwell)1) was added to the cells. After 20 h incubation, for evaluation of viable cell number, the cells were further incubated for 2 h in the presence of 0.25 mM WST-1 and 10 lM 1-methoxy PMS and the absorbance was determined at 450 nm [14]. In order to avoid overestimation, we calculated the concentration of RNase T1 in culture medium with RNase T1/HVJ added on the assumption that all RNase T1 was incorporated in the HVJ envelope vector.

Analysis of DNA fragmentation

Recombinant RNase T1 was purchased from MoBiTec (Go¨ ttingen, Germany). HVJ envelope vector (GenomONE Kit) was from Ishihara Sangyo (Osaka, Japan). The cell proliferation reagent 2-(4-iodophenyl)-3-(4-nitrophenyl)- 5-(2,4-disulphophenyl)-2H-tetrazolium, monosodium salt (WST-1) and 1-methoxy-5-methylphenazinum methylsul- fate (1-methoxy PMS) were obtained from Wako Pure Chemical (Osaka, Japan). Cell culture media and fetal bovine serum (FBS) were from Sigma (St. Louis, MO, USA). The caspase inhibitor z-VAD-fmk was purchased from CN Biosciences (Darmstadt, Germany). All chemicals used in this study were of highest grade commercially available.

Cell culture

Internucleosomal DNA fragmentation was analyzed by agarose gel electrophoresis [15]. K-562 cell culture (1.6 mL) from each experimental condition was centrifuged at 2000 g for 5 min. After the supernatant was removed, the cells were resuspended in 200 lL of 10 mM Tris/HCl, pH 7.6, 1 mM EDTA, 0.2% (v/v) Triton X-100. Following incubation at 37 (cid:2)C for 30min, the nuclei were removed by centrifugation at 10 000 g for 20 min, and the resulting supernatant was treated for 1 h at 55 (cid:2)C with 0.1 mgÆmL)1 RNase A, followed by an additional 1 h of incubation in the presence of 0.3 mgÆmL)1 proteinase K. After two extractions with phenol/chloroform, the fragmentated DNA in the solution was precipitated, resuspended in Tris/EDTA and analyzed by electrophoresis on 2% agarose gel.

Results

K-562, a continuous human chronic myelogenous leukemia cell line, and SAS, a poorly differentiated human squamous cell carcinoma line, were kindly provided by Y. Kaneda of Osaka University (Osaka, Japan). G-402, a human renal leiomyoblastoma cell line, and BHK-21 (C-13), a baby hamster kidney cell line, were purchased from Dainippon Pharmaceutical (Osaka, Japan). K-562 cells were cultured in RPMI1640 medium containing 10% (v/v) FBS. SAS, G-402 and BHK-21 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) FBS. All cells were grown at 37 (cid:2)C in a humidified atmosphere of 5% CO2. Cytotoxic effect of RNase T1 incorporated in HVJ envelope vector

Incorporation of RNase T1 or BSA into HVJ envelope vector

Recently, Kaneda et al. developed a simple method of introducing plasmid DNA into inactivated HVJ particles and delivering the plasmid DNA into various cultured cells and animal tissues [11]. In this method, plasmid DNA was incorporated into HVJ envelope vector by treatment with mild detergent and centrifugation. Therefore, we attempted the introduction of RNase T1 into HVJ envelope vector according to their method and investigated its cytotoxity for K-562 cells.

Thirty microliters of HVJ envelope vector suspension (0.75 assay units) was centrifuged at 10 000 g for 5 min. The supernatant was removed and the pellet was resuspended in RNase T1 or BSA solution (15 lL). After addition of 2% (v/v) Triton X-100 solution (1.5 lL), the mixture was centrifuged at 10 000 g for 5 min. The supernatant was removed and finally the pellet was resuspended in 30 lL of NaCl/Pi. The RNase T1 incorporated in HVJ envelope vector was stored at 4 (cid:2)C until use.

Incorporation of RNase T1 into HVJ envelope vector pretreated with protamine sulfate

To examine the effect of HVJ envelope vector, the cells were treated with RNase T1 alone, HVJ envelope vector alone or RNase T1 incorporated in HVJ envelope vector (RNase T1/HVJ), and 20 h later the surviving cell number was measured using the WST-1 assay [14]. RNase T1/HVJ was clearly more cytotoxic than RNase T1 alone or HVJ envelope vector alone (Fig. 1A), and its cytotoxicity was dependent on the dose of RNase T1 (Fig. 1B), suggesting that HVJ envelope vector enhanced the internalization of RNase T1.

Although HVJ envelope vector alone decreased the cell number slightly, the shape of the cells was indistinguishable from that of untreated cells and different from that of the cells treated with RNase T1/HVJ. In the cell culture treated with RNase T1/HVJ, many cells that had been killed were observed (data not shown). Thirty microliters of HVJ envelope vector suspension (0.75 assay units) was mixed with 7.5 lL of protamine sulfate solution and stored on ice for 5 min. After addition of RNase T1 solution (15 lL) and 2% (v/v) Triton X-100 solution (5.25 lL), the mixture was centrifuged at 10 000 g for 5 min. The supernatant was removed and finally the pellet was resuspended in 30 lL of NaCl/Pi. The RNase T1 incorporated in HVJ envelope vector was stored at 4 (cid:2)C until use.

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Fig. 1. Effect of incorporation in HVJ envelope vector on the cytotoxi- city of RNase T1. (A) K-562 cells were incubated for 20 h at 37 (cid:2)C in the presence of BSA, RNase T1, HVJ envelope vector, BSA incor- porated in HVJ envelope vector (BSA/HVJ) or RNase T1 incorpor- ated in HVJ envelope vector (RNase T1/HVJ). In all cell cultures containing either BSA or RNase T1, their concentrations were 10 lgÆmL)1. (B) K-562 cells were incubated for 20 h at 37 (cid:2)C in the presence of different concentrations of RNase T1 incorporated in HVJ envelope vector. Viable cell number was measured by WST-1 assay as described in Experimental procedures. Each point and bar represents the mean value ± SD of cell number as percentage of attenuance in control cultures.

Fig. 2. Effect of pretreatment of HVJ envelope vector with protamine sulfate on the cytotoxicity of RNase T1/HVJ. (A) K-562 cells were incubated for 20 h at 37 (cid:2)C in the presence of RNase T1 treated with 0.2 mgÆmL)1 protamine sulfate (RNase T1/PS), HVJ envelope vector treated with 0.2 mgÆmL)1 protamine sulfate (PS/HVJ), RNase T1 incorporated in HVJ envelope vector (RNase T1/HVJ) or RNase T1 incorporated in HVJ envelope vector pretreated with 0.2 mgÆmL)1 protamine sulfate (RNase T1/PS/HVJ). In all cell cultures containing RNase T1, its concentration was 1 lgÆmL)1. (B) K-562 cells were incubated for 20 h at 37 (cid:2)C in the presence of 0.1 lgÆmL)1 RNase T1 incorporated in HVJ envelope vector pretreated with different con- centrations of protamine sulfate (black bars) or HVJ envelope vector treated with different concentrations of protamine sulfate (hatched bars). Viable cell number was measured by WST-1 assay as described in Experimental procedures. Each bar represents the mean value ± SD of cell number as percentage of attenuance in control cultures.

When BSA was used instead of RNase T1, BSA incor- porated in HVJ envelope vector (BSA/HVJ) was slightly more cytotoxic than BSA alone. However, the surviving cell number with BSA/HVJ treatment was nearly equal to that of treatment with HVJ alone (Fig. 1A). We therefore suspected that the effect of BSA/HVJ was due to HVJ while the cytotoxicity of RNase T1/HVJ was mainly due to ribonuclease activity of RNase T1 internalized via the HVJ envelope vector.

Effect of protamine sulfate on incorporation of RNase T1 in HVJ envelope vector not shown). Therefore, we tested its effect on incorporation of RNase T1.

As shown in Fig. 2A, pretreatment with protamine sulfate drastically enhanced cytotoxicity (RNase T1/HVJ vs. RNase T1/PS/HVJ). Because RNase T1 plus protamine In our review of Kaneda’s method, we found that the pretreatment of HVJ envelope vector with protamine sulfate enhanced incorporation of plasmid DNA in the vector (data

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(PS/HVJ) did not cause cell death,

baby hamster kidney cell line BHK-21 (C-13). These cell lines were as sensitive as K-562 and SAS (data not shown). Unfortunately, cytotoxicity specific to cancer cells was not observed, unlike previous findings for onconase [1] and bovine seminal RNase [2].

Mode of cell death induced by RNase T1/PS/HVJ

sulfate without HVJ envelope vector (RNase T1/PS) or HVJ envelope vector alone with protamine sulfate pre- treatment the observed cytotoxicity had undoubtedly been induced by the RNase T1 incorporated in or associated with the vector. Next, we investigated the effects of various concentra- tions of protamine sulfate in the pretreatment of HVJ envelope vector (Fig. 2B). The cytotoxicity of RNase T1/ HVJ increased in proportion to the concentration of protamine sulfate up to 0.6 mgÆmL)1.

Anticancer drugs induce cell death via apoptosis or necrosis. Because necrotic cell death could produce severe inflamma- tory and immune complications in patients, necrosis could hamper application to the treatment of cancer. On the other hand, apoptosis induces weaker responses in the body. Cytotoxicity of optimized RNase T1/HVJ (RNase T1/PS/ HVJ)

Apoptosis is defined by characteristic morphological changes associated with digestion of chromatin, a process that can be visualized as an oligonucleosome-sized DNA ladder on an ethidium bromide-stained agarose gel [16]. Soluble (fragmented) DNA was isolated from control, HVJ envelope vector-treated and RNase T1/PS/HVJ-treated K-562 cells and analyzed on agarose gels (Fig. 4). Only

RNase T1 incorporated in the HVJ envelope vector pre- treated with 0.6 mgÆmL)1 protamine sulfate (RNase T1/PS/ HVJ) was assayed for cytotoxicity on two types of human tumor cell lines: human erythroleukemia cell line K-562 and human tongue carcinoma cell line SAS. First, we examined the cytotoxic effect of RNase T1 alone on these tumor cells. Although at the highest concentration (100 lgÆmL)1) the number of K-562 cells was slightly diminished, RNase T1 alone had no other effect on K-562 or SAS cell viability at the concentration used in this assay (Fig. 3). On the other hand, as shown in Fig. 3, both human tumor cell lines were very sensitive to RNase T1/PS/HVJ, resulting in enhance- ment of the cytotoxicity of RNase T1 by several orders of magnitude. RNase T1 cytotoxicity (IC50 > 100 lgÆmL)1) was increased at least 1000-fold when RNase T1 was incorporated in the HVJ envelope vector pretreated with protamine sulfate (IC50 ¼ 0.1 lgÆmL)1). In this experiment, tumor cells were incubated with RNase T1 for 20 h, because longer incubation (48 or 72 h) increased its cytotoxicity only minimally.

Fig. 4. Detection of internucleosomal DNA fragmentation in K-562 cells induced by RNase T1/PS/HVJ. K-562 cells were incubated in the presence of HVJ envelope vector treated with 0.6 mgÆmL)1 protamine sulfate (PS/HVJ) or 1 lgÆmL)1 RNase T1 incorporated in HVJ envelope vector pretreated with 0.6 mgÆmL)1 protamine sulfate (RNase T1/PS/HVJ). Twenty hours later, cells were harvested and their fragmented DNA was analyzed as described in Experimental procedures. Lane 1, molecular mass marker (k HindIII); lane 2, untreated control; lane 3, PS/HVJ; lane 4, RNase T1/PS/HVJ; lane 5, molecular mass marker (100 bp ladder).

Fig. 3. Cytotoxic effect of RNase T1/PS/HVJ in human tumor cell lines. K-562 cells (circles) or SAS cells (squares) were incubated for 20 h at 37 (cid:2)C in the presence of different concentrations of RNase T1 (open symbols) or RNase T1 incorporated in the HVJ envelope vector pretreated with 0.6 mgÆmL)1 protamine sulfate (closed symbols). Viable cell number was measured by WST-1 assay as described in Experimental procedures. Each point represents the mean value ± SD of cell number as percentage of attenuance in control cultures.

We studied the effect of RNase T1/PS/HVJ on two more cell lines: human renal leiomyoblastoma cell line G-402 and

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Fig. 5. Effect of the caspase inhibitor z-VAD-fmk on the cytotoxicity of RNase T1/PS/HVJ. K-562 cells were incubated for 20 h with HVJ envelope vector treated with 0.6 mgÆmL)1 protamine sulfate (PS/HVJ) or 1 lgÆmL)1 RNase T1 incorporated in HVJ envelope vector pre- treated with 0.6 mgÆmL)1 protamine sulfate (RNase T1/PS/HVJ) in either the absence (hatched bars) or presence (black bars) of 50 lM z-VAD-fmk. Viable cell number was measured by WST-1 assay as described in Experimental procedures. Each bar represents the mean value ± SD of cell number as percentage of attenuance in control cultures.

origin of the cell line, as the K-562 human erythroleukemia cell line and the SAS human tongue carcinoma cell line exhibited the same IC50 value. However, it was not specifically toxic to tumor cells, unlike onconase [1] and bovine seminal RNase [2], because the BHK-21 baby hamster kidney cell line was also sensitive to it.

RNase T1/PS/HVJ-treated cells displayed the characteris- tic internucleosomal chromatin cleavage indicating that RNase T1 triggered apoptotic cell death programs.

Pretreatment with protamine sulfate drastically increased the cytotoxicity of RNase T1/HVJ (Fig. 2). Protamine sulfate has been used widely to enhance gene transfer. Gao & Huang reported that several high molecular mass cationic polymers, including protamine, enhanced the transfection efficiency of several types of cationic liposomes in vitro because of the formation of favorable structures of highly condensed, lipid-associated particles [22]. On the other hand, Kaneda et al. speculated that the low transfec- tion efficiency of HVJ envelope vector resulted from weak association of the vector with the cell membrane due to the negative charge on both the envelope and the cell mem- brane. They therefore used protamine sulfate to augment attachment of the HVJ envelope vector to the cell surface by providing a cationic charge on the HVJ surface [11]. Furthermore, we observed that pretreatment of the HVJ envelope vector with protamine sulfate enhanced incorpor- ation of plasmid DNA in the vector (data not shown). The above findings suggest several possible explanations of the potentiation by protamine sulfate of the cytotoxicity of RNase T1/HVJ. First, because RNase T1 is an acidic protein, protamine forms a condensed complex with RNase T1, and enhances attachment of RNase T1 to the negatively charged surface of HVJ envelope vector, as a result of which incorporation of RNase T1 into the HVJ envelope vector is augmented. Secondly, as Kaneda et al. pointed out, protamine accelerates attachment of the HVJ envelope vector to the cell surface [11].

Caspases (cysteine aspartate-specific proteases) play crit- ical roles in the control and induction of the apoptotic cascade [17]. Actually, many types of apoptotic cell death are inhibited by the cell-permeable irreversible caspase inhibitor z-VAD-fmk [18–20]. As shown in Fig. 5, pretreat- ment with 50 lM z-VAD-fmk, partially, but not completely, rescued K-562 cells from death induced by RNase T1/PS/ HVJ. This result suggested that RNase T1/PS/HVJ induced cell death via both apoptotic (z-VAD-fmk-sensitive) and nonapoptotic (z-VAD-fmk-resistant) pathways.

Discussion

In our preliminary examination, we observed that besides RNase T1, several proteins (BSA, IgG, lysozyme and b-galactosidase) were internalized into cells by transfection using HVJ envelope vector pretreated with protamine sulfate. We therefore believe that HVJ envelope vector will be useful as a novel tool for protein transfection, although it is uncertain whether it will be useful for all proteins, in particular those that are intrinsically capable of being internalized into cells and/or those that are cationic such as onconase or bovine seminal RNase.

The typical apoptosis-related DNA ladder was detected in the electrophoretic analysis of DNA from cells incubated with RNase T1/PS/HVJ (Fig. 4). Moreover, the caspase inhibitor z-VAD-fmk significantly protected cells from RNase T1/PS/HVJ-induced cell death (Fig. 5). These results suggest that RNase T1/PS/HVJ can induce apopto- sis in K-562 cells. The K-562 cell line is derived from a chronic myelogenous leukemia (CML) patient and expres- ses the BCR-ABL fusion protein [23]. This aberrant expression of the abl oncogene renders K-562 cells resistant to apoptotic cell death induced by anticancer drugs [24]. BCR-ABL expression is of fundamental pathogenic import- ance in CML [25,26]. RNase T1/PS/HVJ might be useful for the treatment of CML.

Cytotoxic RNases are expected to be useful as nonmuta- genic alternatives to the conventional DNA-damaging therapy of cancer [1]. The purpose of this study was to develop a new method of efficiently delivering RNases to cancer cells by utilizing a novel gene transfection reagent, HVJ envelope vector. As a model RNase, we selected RNase T1, which was considered noncytotoxic because of its inability to translocate across the plasma membrane. RNase T1 was found not to be significantly cytotoxic to either K-562 cells or SAS cells (IC50 > 100 lgÆmL)1; Fig. 3). On the other hand, incorporation of RNase T1 in the HVJ envelope vector pretreated with protamine sulfate times (IC50 ¼ increased its cytotoxicity thousands of 0.1 lgÆmL)1 ¼ 0.01 lM; Fig. 3). This cytotoxicity was stronger than the reported cytotoxicity (0.4 lM) of onconase [21], which is currently in clinical trials as a cancer chemotherapeutic agent. Moreover, the cytotoxicity of RNase T1/PS/HVJ does not appear to be related to the It was reported that onconase or a-sarcin also triggered the apoptotic cell death program [5,9]. Although these ribonucleases have different substrate specificities (onconase preferentially degrades tRNA in HeLa cells and a-sarcin

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evidence for cytotoxic mechanisms different from inhibition of protein synthesis. Cancer Res. 60, 1983–1994.

10. Bracale, A., Spalletti-Cernia, D., Mastronicola, M., Castaldi, F., Mannucci, R., Nitsch, L. & D’Alessio, G. (2002) Essential stations in the intracellular pathway of cytotoxic bovine seminal ribonuc- lease. Biochem. J. 362, 553–560.

11. Kaneda, Y., Nakajima, T., Nishikawa, T., Yamamoto, S., Ike- gami, H., Suzuki, N., Nakamura, H., Morishita, R. & Kotani, H. (2002) Hemagglutinating virus of Japan (HVJ) envelope vector as a versatile gene delivery system. Mol. Ther. 6, 219–226.

12. Yoshida, H. (2001) The ribonuclease T1 family. In Methods in Enzymology, Vol. 341 (Nicholson, A.W., ed), pp. 28–41. Academic Press, New York.

13. Mariani, C., De Beuckeleer, M., Truettner, J., Leemans, J. & Goldberg, R.B. (1990) Induction of male sterility in plants by a chimeric ribonuclease gene. Nature 347, 737–741.

cleaves 28S rRNA at a specific site in rhabdmyosarcoma cells), it is clear that they cause cell death by degrading RNA [5,9]. Although we did not evaluate the ribonuclease activity of internalized RNase T1, it may degrade certain RNAs commonly digested by cytotoxic ribonucleases. Alternat- ively, because some toxins, which inhibit protein biosyn- thesis, induce apoptosis [27], RNase T1 may trigger the apoptotic cell death pathway via inhibition of protein biosynthesis.

14. Ishiyama, M., Shiga, M., Sasamoto, K., Mizoguchi, M. & He, P. (1993) A new sulfonated tetrazolium salt that produces a highly water-soluble formazan dye. Chem. Pharm. Bull. 41, 1118–1122. 15. Pe´ rez-Sala, D. & Mollinedo, F. (1994) Inhibition of isoprenoid biosynthesis induces apoptosis in human promyelocytic HL-60 cells. Biochem. Biophys. Res. Commun. 199, 1209–1215.

16. Gerschenson, L.E. & Rotello, R.J. (1992) Apoptosis: a different

In this study, we demonstrated that a novel gene transfer reagent, HVJ envelope vector, makes the noncytotoxic RNase, RNase T1, cytotoxic to cancer cells. This cytotox- icity is stronger than that of onconase. Furthermore, this RNase T1 induces the apoptotic cell death program. Because its cytotoxicity is not specific to tumor cells, it cannot at present be developed as an anticancer drug. However, it should be a unique anticancer drug if HVJ envelope vector can be targeted to tumor cells. A study of this strategy is currently in progress.

Acknowledgements

type cell death. FASEB J. 6, 2450–2455.

17. Thornberry, N.A. & Lazebnik, Y. (1998) Caspases: enemies

within. Science 281, 1312–1316.

We sincerely thank Dr Yasufumi Kaneda for his useful guidance and supplying the human tumor cell lines. We also thank Ms Mika Tanaka and Mr Takaharu Yagi for their experimental assistance.

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