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

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 Several ribonucleases, i ncluding onconase and a-sarcin, are known t o be t oxic 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 c ells. In this s tudy, we internalized RNase T 1 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 su lfate, and was stronger than that of onconase, which i s i n phase III human clinical trials as a nonmutagenic cancer chemothe rapeutic agent. Furthermore, internalized RNase T1 induced apoptotic cell death p rograms. Because its c ytotoxicity 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: a poptosis; HVJ envelope vector; protein trans- fection; RNase T1; tumor cell death. Ribonucleases are b est known f or their ability to cleave RNA. However, some of these proteins have been shown t o be much more than digestive enzymes. For example, two groups of these e nzymes have antitumor a ctivities [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 chemotherapeu tic agent i n phase III clinical trials for mesothelioma [1]. 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 t he target cell and enters it by endocytosis [6]. They u ndergo internalization intrinsically, but their translocation across the plasma membrane is the rate-limiting step of the cytotoxic effe ct [6]. Therefore, many research groups have created fusion proteins to enhance the interactions between RNases and the plasma me mbrane [7]. T he 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 L ipofectin-mediated d elivery o f onc onase, t hough they did not aim at its clinical use [9]. In order to k ill cells, internalized RNase must reach the cytosol, where RNA exists. Bovine seminal RNase, a dimeric RNase selectively cytotoxic for malignant cells, i s 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. 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 w ith the plasma membrane of target cells, the plasmid DNA is easily and directly introduced into the c ytosol without degradation by e ndo- somes a nd lysosomes. Th erefore, we expected that the HVJ envelope vector trapped RNase could b ecome an effective antitumor agent. RNase T1 from the fungus Aspergillus o ryzae is a small acidic protein structurally related to a-sarcin but nontoxic to tumor cells [12]. The tap etum 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 e fficient introduction into target cells could cause their death. We therefore considered RNas e T1 a good model for evaluating the effect 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 200 4, revised 30 J une 2004, accepted 1 9 July 2004) Eur. J. Biochem. 271, 3567–3572 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04293.x of HVJ envelope vector on RNase cytotoxicity, prep ared RNase T1-containing HVJ envelope vector and investi- gated its cytotoxicity for tumor cell lines. Experimental procedures Materials Recombinant RNase T1 was purchased from MoBiTec (Go ¨ ttingen, Germany). HVJ envelope vector (GenomON E 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) w ere 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 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. K aneda 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. S AS, G-402 and BHK-21 cells were cultured in Dulbecco’s modified Eagle’s m edium supplemented w ith 10% (v/v) FBS. All cells were grown at 37 °C in a humidified atmosphere of 5% CO 2 . Incorporation of RNase T1 or BSA into HVJ envelope vector Thirty microliters o f HVJ envelope vector suspension (0.75 assay units) was centrifuged at 10 000 g fo r 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 3 0 lLof NaCl/P i . The RNase T1 incorporated in HVJ envelope vector was stored at 4 °C until use. Incorporation of RNase T1 into HVJ envelope vector pretreated with protamine sulfate Thirty microliters o f HVJ envelope vector suspension (0.75 assay units) was mixed with 7.5 lL of protamine sulfate solution and s tored on i ce for 5 min. After addition of RNase T1 s olution (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 lLofNaCl/P i .TheRNaseT1 incorporated in HVJ envelope vector was stored at 4 °C until use. Cytotoxicity assay K-562 (1 · 10 4 cells per well) or SAS (4 · 10 3 cells per well) cells were seeded in a 96 well p late. F ollowing 24 h i ncubation 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 m M WST-1 a nd 10 l M 1-methoxy PMS and the absor bance was determined at 450 nm [14]. In order to avoid overestimation, we calculated the concentration o f R Nase T1 in culture medium with RNase T1/HVJ a dded on t he assumption that all RNase T1 was incorporated in the HVJ envelope vector. Analysis of DNA fragmentation Internucleosomal DNA fragmentation was analyzed by agarose gel electrophore sis [15]. K-562 cell c ulture (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 lLof10m M Tris/HCl, pH 7.6, 1 m M EDTA, 0 .2% (v/v) Triton X-100. Following incubation at 37 °C for 30min, the nuclei were removed by centrifugation at 10 000 g for 20 min, a nd the resulting supernatant was treated for 1 h at 55 °C with 0.1 mgÆmL )1 RNase A, followed b y 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 Cytotoxic effect of RNase T1 incorporated in HVJ envelope vector Recently, Kaneda et al. developed a simple method of introducing plasmid DNA into inactivated HVJ particles and d elivering t he 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. To examine t he effect o f 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 late r the surviving cell number was measured u sing 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 t he 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). 3568 S. Yuki et al.(Eur. J. Biochem. 271) Ó FEBS 2004 When BSA w as used instead of RNase T1, BSA incor- porated in HVJ envelope vector (BSA/HVJ) was slightly more cytotoxic than B SA alone. H owever, the surviving cell number with BSA/HVJ treatmen t was nearly equal t o that of treatment with HVJ alone (Fig. 1 A). 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 a ctivity of RNase T1 internalized via the HVJ envelope vector. Effect of protamine sulfate on incorporation of RNase T1 in HVJ envelope vector In our review of Kaneda’s method, we fou nd that the pretreatment of HVJ envelope vector with protamine sulfate enhanced incorporation of plasmid DNA in the vector (data not shown). Therefore, we tested i ts effect on incorporation of RNase T 1. As shown in Fig. 2A, pretreatment with protamine sulfate drastically enhanced cytotoxicity (RNase T1/HVJ vs. RNase T1/PS/HVJ). B ecause R Nase T1 plus protamine Fig. 1. Effect of incorporation i n HVJ envelope vector o n the cytotoxi- city of RNa se T1. (A) K-562 cells were incubated for 20 h at 37 °Cin the presence of BSA, R Nase T1, HVJ en velope vector, BSA inc or- porated in HVJ envelope vector (BSA/HVJ) or R Nase T1 incorpor- ated in HVJ e nvelope vector (RNase T1/HVJ). In all cell cu ltures containing either BSA or RNase T1, their concentrations were 10 lgÆmL )1 . (B) K-562 cells were incubated for 20 h at 3 7 °Cinthe presence of different concentrations of R Nase T1 incorporated i n HVJ envelope vec tor. Viable cell num ber w as measured by WST -1 assay as described in Experimental p rocedu res. Each point a nd bar represents the m ean value ± SD of cell nu mber as perce ntage of attenuan ce in control cultures. Fig. 2. Eff ec t of p retrea tment o f HVJ e nve lope ve ctor with protamine sulfate on the cytotoxicity of RNase T1/HVJ. (A) K-562 cells were incubated for 20 h at 37 °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/H VJ), 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 c ultures containing RNase T1, its concentratio n was 1 lgÆmL )1 . (B) K-562 cells were incubated for 20 h at 37 °C in t he presence of 0.1 lgÆmL )1 RNase T1 incorporated in HVJ envelope vector pretreated with d ifferent con- centrations of protamine sulfate (black bars) or HVJ envelope vector treated with different concentrations of protamine sulfate (hatched bars). Viable cell numbe r was measured by WST - 1 assay as describe d in Experimental procedures. Each bar represents the mean value ± SD of cell number as percentage of attenuance in c ontro l cultures. Ó FEBS 2004 Cytotoxicity of RNase T1 via HVJ envelope vector (Eur. J. Biochem. 271) 3569 sulfate without HVJ envelope vector (RNase T1/PS) or HVJ envelope vector alone with protamine s ulfate pre- treatment (PS/HVJ) did not cause cell death, the observed cytotoxicity had undoubtedly been induced by the RNase T1 incorporated in or asso ciated with the vector. Next, we investigated the effects of various concentra- tions of protamine s ulfate 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 . Cytotoxicity of optimized RNase T1/HVJ (RNase T1/PS/ HVJ) RNase T1 incorporated in the HVJ envelope vector pre- treatedwith0.6 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 to ngue carcinoma cell line SAS. First, we examined the cytotoxic effect of RNase T1 alone on these t umor 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 o r SAS cell viability a t the concentratio n used in this a ssay ( Fig. 3). On the other hand, as shown i n 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 (IC 50 >100lgÆmL )1 ) was increased at least 1000-fold when RNase T1 was incorporated in the HVJ envelope vector pretreated with protamine su lfate (IC 50 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. We studied the effect of RNase T1/PS/HVJ on two more cell lines: human renal leiomyoblastoma cell line G-402 and baby hamster kidney cell line BHK-21 (C-13). These cell lines were as sensitive as K-562 and SAS (data n ot shown). Unfortunately, c ytotoxicity 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 Anticancer drugs induce cell death via apoptosis or necrosis. Because necrotic cell death cou ld produce severe inflamma- tory and i mmune complications in patients, necrosis could hamper application t o the tr eatment o f cancer. O n t he other hand, apoptosis induces weaker responses in the body. Apoptosis is defined by characteristic morphological changes associated with digestion of chromatin, a process that can be visualized as an oligonucleosome-sized DNA ladder o n an ethidium bromide-stained agarose g el [16]. Soluble (fragmented) DNA was isolated from control, HVJ envelope vector-treated and RNase T1/PS/HV J-treated K-562 cells and analyzed on agarose gels (Fig. 4). Only 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 a t 37 °C in t he presence of different conce ntrations of RNase T1 (open s ymbols) or RNase T1 incorporated in the HVJ envelope ve ctor pretreated with 0.6 mgÆmL )1 protamine sulfate (closed s ymbols). Viable cell number was measured by WST-1 assay as described in Experimental procedures. Each p o int represents the mean valu e ± SD of cell number as percentage of attenuance in control cultures. Fig. 4. Detection of internucleosomal DNA fragm entation in K-562 cells induced by RNase T1/PS/HVJ. K-562cellswereincubatedinthe presence of HVJ envelope vector t reated with 0.6 mgÆmL )1 protamine sulfate (PS/HVJ) or 1 lgÆmL )1 RNase T 1 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 D NA was analyzed as described in Experimental procedures. Lane 1, molecular mass marker (k HindIII); lane 2, untreated co ntrol; l ane 3 , P S/HVJ; lane 4, RNase T1/PS/H VJ; l ane 5 , molecular mass marker (100 bp ladder). 3570 S. Yuki et al.(Eur. J. Biochem. 271) Ó FEBS 2004 RNase T1/PS/HVJ-treated cells displayed the characteris- tic internucleosomal chromatin cleavage indicating that RNase T1 triggered a poptotic cell death programs. Caspases (cysteine aspartate-specific proteases) play crit- ical roles in the control a nd 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 l M z-VAD-fmk, partially, but not completely, rescued K-562 cells from death induced by RNase T1/PS/ HVJ. This result suggested that R Nase T1/PS/HVJ induced cell death via both apoptotic (z-VAD-fmk-sensitive) and nonapoptotic (z-VAD-fmk-resistant) pathways. Discussion 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 b e significantly cytotoxic to either K-562 cells or SAS cells (IC 50 > 100 lgÆmL )1 ; Fig. 3). On the other hand, incorporation of RNase T1 in the HVJ envelope vector pretreated with protamine sulfate increased its cytotoxicity thousands of times (IC 50 ¼ 0.1 lgÆmL )1 ¼ 0.01 l M ; Fig. 3 ). This cytotoxicity was stronger than the reported cytotoxicity (0.4 l M ) o f 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 origin of the cell line, as the K-562 human erythroleukemia cell line and the SAS human tongue carcinoma cell line exhibited the same IC 50 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. Pretreatment with protamine s ulfate drastically increased the cytotoxicity of RNase T1/HVJ (Fig. 2). Protamine sulfate has been used widely to en hance gene transfer. Gao & Huang reported t hat 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 o f the vecto r 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 T 1 to the negatively charged surface of HVJ envelope vector, as a result of which i ncorporation of RNase T1 into the HVJ envelope vector is augmented. Secondly, as Kaneda et al. pointed out, protamine accelerates attachment of t he HVJ envelope vector to the cell surface [11]. In our pr eliminary 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 HV J envelope vector will be useful as a novel tool for p rotein 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 t hat are cationic such as onconase or bovine seminal RNase. The typical apoptosis-related DNA ladder was d etected in the electrophoretic analysis of DNA fro m cells incubated with RNase T1/PS/HVJ (Fig. 4). More over, the caspase inhibitor z-VAD-fmk significantly protected cells from RNase T1/PS/HVJ-induced cell d eath (Fig. 5). These results suggest that RNase T1/PS/HVJ can i nduce apopto- sis in K-562 cells. The K-562 c ell 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. It was reported that onconase or a-sarcin also triggered the apoptotic cell death program [5,9]. Although these ribonucleases have different substrate s pecificities (oncon ase preferentially degrades tRNA in HeLa cells and a-sarcin Fig. 5. Effect of t he caspase inhibitor z-VAD-fmk on the cytotox icity 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 i ncorporate d in HVJ en velope ve ctor p re- treated w ith 0.6 mgÆmL )1 protamine s ulfate (RNase T1/PS/HVJ) in either the absence (hatched bars) o r presence ( black bars) of 50 l M z-VAD-fmk. Viable ce ll n umber was measured by WST-1 a ssay a s described in Experimental p ro cedure s. E ach bar represents the mean value ± SD of cell number as percentage of attenuance in control cultures. Ó FEBS 2004 Cytotoxicity of RNase T1 via HVJ envelope vector (Eur. J. Biochem. 271) 3571 cleaves 28S rRNA at a specific site in rhabdmyosarcoma cells), it is clear that t hey cause cell death by d egrading RNA [5,9]. Although w e did not evalua te the r ibonuclease a ctivity 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], R Nase T1 may trigger the apoptotic cell death pathway via inhibition of protein biosynthesis. 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 i s s tronger than that of onconase. Furthermore, this RNase T1 induces the apoptotic cell death program. Because its cytotoxicity is not specific to tumor c ells, it cannot at present b e developed a s an anticancer drug. 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