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:
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.
However, it should be a unique anticancer drug if HVJ
envelope vector can be t argeted to tumor cells. A study of
this strategy is currently in progress.
Acknowledgements
We sin cerely thank D r Yasufumi K aneda for h is useful guidance and
supplying the huma n tumor cel l lines. W e also thank Ms Mika Tanaka
and Mr Takaharu Yagi for their e xperimenta l assistance.
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