Báo cáo Y học: Mistletoe viscotoxins increase natural killer cell-mediated cytotoxicity pptx

Mistletoe viscotoxins increase natural killer cell-mediated
cytotoxicity
Julie Tabiasco
1
, Fre
´
de
´
ric Pont
2
, Jean-Jacques Fournie
´
1
and Alain Vercellone
1
1
Institut National de la Sante
´
et de la Recherche Me
´
dicale U563 and
2
Service de spectrome
´
trie de masse de l¢ IFR 30,
CHU Purpan, BP3028, Toulouse, France
Mistletoe extracts have immunomodulatory activity. We
show that nontoxic concentrations of Viscum album extracts
increase natural killer (NK) cell-mediated killing of tumor
cells but spare nontarget cells from NK lysis. The com-
pounds responsible for this bioactivity were isolated from

mistletoe and characterized. They have low molecular mass
and are thermostable and protease-resistant. After complete
purification by HPLC, they were identified by tandem MS as
viscotoxins A1, A2 and A3 (VTA1, VTA2 and VTA3,
respectively). Whereas micromolar concentrations of these
viscotoxins are cytotoxic to the targets, the bioactivity with
respect to NK lysis is within the nanomolar range and differs
between viscotoxin isoforms: VTA1 (85 n
M
), VTA2 (18 n
M
)
and VTA3 (8 n
M
). Microphysiometry and assays of cell
killing indicate that, within such nontoxic concentrations,
viscotoxins do not activate NK cells, but act on cell conju-
gates to increase the resulting lysis.
Keywords: mistletoe; natural killer cells; tumor cells; visco-
toxin; Viscum album.
Mistletoe preparations have been used for pharmacological
purposes since ancient times [1]. These days, industrially
produced extracts from mistletoe are used in treatments of
solid tumors [2–5]. It is thought that the molecular basis of
the antitumoral activity of mistletoe lies in several distinct
bioactivities. First, its lectin content is responsible for direct
toxicity to tumor cells [6–9]. Secondly, the Viscum album
rhamnogalacturonan oligosaccharide favors bridging of
natural killer (NK)–tumor cell conjugates, enhancing effi-
ciency of killing [10–15]. Thirdly, it has been found that the

antitumoral human cytotoxic T lymphocytes with cd Tcell
receptor are selectively activated by mistletoe ligands of
phosphoantigen structure [16,17].
NK cells play an important role in antitumoral immunity
as they directly kill tumor cells and regulate the adaptative
immunity [18]. Activation of cytolytic functions of NK cells
relies mainly on selective interactions of NK receptors with
major histocompatibility complex (MHC) class I-related
molecules on the tumoral cells. Distinct ligands, however,
may also induce NK cell stimulation [19–23]. Hence,
immunotherapeutic modulation of NK cell activation is
an important issue in current antitumoral approaches.
We analysed the bioactivity of V. album compounds in
the in vitro killing of tumor cells by human NK cells and
found that the components that mediate this bioactivity are
viscotoxins.
MATERIALS AND METHODS
Preparation of
V. album
(Va) extract
The green and white parts of mistletoe (V. album L.,
Viscaceae; 1 kg) freshly collected on Robinia pseudacacia L.
were crushed and extracted twice with 5 L methanol/water
(1 : 1, v/v). After filtration and volume reduction to
600 mL, the aqueous phase was successively partitioned
with cyclohexane, dichloromethane and ethyl acetate. This
aqueous phase was positive in tests of enhancement of
NK-mediated killing of tumor cells, but was directly toxic
for the tumor target cell line. Ethanol was added to the
concentrated aqueous phase to achieve 85% (v/v) concen-

tration. A precipitate was obtained and separated from the
supernatant by centrifugation (2000 g; 10 min). The super-
natant was concentrated and ethanol was added to 85%
(v/v). After centrifugation, the precipitate was dissolved in
water, pooled with the former precipitate, and constituted
the Va extract. The final yield of Va extract was 50 g from
1 kg plant extracted. The stock solution of Va extract
(237 mgÆmL
)1
) was stored at )20 °C.
Viscotoxins
The protocol for purifying viscotoxins was modified from a
previous method [24,25]. Briefly, to fractionate Va extract,
we replaced ion-exchange and exclusion chromatography
with C
18
reverse-phase open column chromatography to
avoid the use of salt solutions. A 4-g portion of Va extract
was applied to a column (2.5 · 20 cm) of Lichroprep RP-18
(Merck, Darmstadt, Germany), which was irrigated with
300 mL 20%, 40% and 100% acetonitrile in 0.1% aqueous
acetic acid. The former 40% acetonitrile eluate was freeze-
dried, dissolved in 20% acetonitrile in 0.1% trifluoroacetic
acid and chromatographed by C
18
reverse-phase HPLC
(Nucleosil 5 lm; 300 A
˚
pore size; Bischoff, Leonberg,
Germany). The column (250 · 4.6 mm) was eluted at

Correspondence to: A. Vercellone, INSERM U395, CHU Purpan,
BP 3028, 31024 Toulouse, France.
Fax: 335 6274 8386, Tel.: 335 6274 8364,
E-mail:
Abbreviations: MHC, major histocompatibility complex; MS
n
,
multiple-stage MS; NK, natural killer; VTA, viscotoxin A; TNF-a,
tumor necrosis factor a.
(Received 28 November 2001, revised 27 March 2002,
accepted 15 April 2002)
Eur. J. Biochem. 269, 2591–2600 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02932.x
1mLÆmin
)1
by a linear gradient from 20% to 50% of
acetonitrile in 0.1% trifluoroacetic acid over 30 min. For
final purification of VTA2 and VTA3, the elution was
carried out with the following gradient: 25% solvent B
(acetonitrile in 0.1% trifluoroacetic acid) and 75% solvent
A (0.1% aqueous trifluoroacetic acid) during the first
10 min, 1 min up to 30% solvent B, 9 min with 30% solvent
B, and from 30% up to 40% over 5 min. Cation-exchange
chromatography was conduced on a polypore SP 10 micron
(100 · 2.1 mm) column (Applied Biosystems) using linear
gradient elution at 0.3 mLÆmin
)1
from 100% solvent A
(50 m
M
phosphate buffer, pH 7) to 100% solvent B (1

M
NaCl in 50 m
M
phosphate buffer, pH 9) in 30 min. The
VTA3 standard was kindly provided by K. Urech and
purified as described by Schaller et al.[24].
Chemical and enzymatic treatments
Dilution in organic solvent was achieved by addition of pure
acetonitrile to 80% final volume. After 2 h at room
temperature, organic solvent was removed by evaporation
with a Speed-vac centrifuge before further bioassays. For
chemical treatments, 1 vol. Va extract or purified viscotoxin
was mixed with 1 vol. 4
M
NaOH or 4
M
HCl and
incubated for 2 h at 37 °C. After neutralization with HCl
or NaOH, the samples were immediately diluted in RPMI
medium supplemented with 10% human serum, and pH
was adjusted to 7.0 before further bioassays. For periodate
oxidation, sodium periodate (Sigma) was added (final
concentration 5 m
M
) to the Va extract or to purified
viscotoxin, and samples were left for 2 h at room tempera-
ture. Unchanged sodium periodate was further neutralized
by the addition of a few drops of glycerol to the sample.
Reduction and alkylation were performed as described
previously [26]. Excess reagent was removed by purification

on a tC
18
September–PakÒ cartridge (Walters, Milford,
MA, USA) eluted successively with increasing percentages
of acetonitrile.
Enzymatic treatments consisted of incubating Va extract
for 2 h at 37 °C with proteinase K (1.5 UÆmL
)1
; Boehringer,
Mannheim, Germany), calf alkaline phosphatase
(1 UÆmL
)1
; Boehringer), or sulfatase (16.8 UÆmL
)1
Aeromonas aerogenes sulfohydrolase; Sigma) in 10 m
M
Tris/HCl, pH 7.2. Heating at 75 °C for 10 min stopped
enzymatic reactions before further bioassay.
SDS/PAGE
SDS/PAGE analysis of Va extract and viscotoxins was
performed using 7 · 10 cm gels of 20% acrylamide (37.5 : 1
ratio of acrylamide to bisacrylamide) for the resolving gel
and 5% acrylamide for the stacking gel. Samples were
electrophoresed at 75 V, and further stained by Coomassie
Blue or silver nitrate.
Mass spectrometry
MS was performed using the LCQ Ion-trap mass spectro-
meter (Thermo-Finnigan, San Jose
´
, CA, USA) by liquid

chromatography/MS and nanospray as described [27]. The
viscotoxin masses were obtained by spectral deconvolution
with Bioworks software from the Xcalibur 1.2 suite
(Thermo-Finnigan). Multiple stage MS (MS
n
) sequencing
of viscotoxins was carried out on the underivatized sample.
The peak at m/z 966.5 (i.e. z ¼ +5forVTA2forwhich
the mass is ¼ 4827 Da) was selected in full-scan MS and
fragmented. In its MS
2
spectrum, Y ions corresponding to
the disulfide-free C-terminal moiety of VTA2 were selected
and fragmented by MS
3
. Fragmentation of the Y
6
ion (m/z
706.3) led to overlapping sets of Y and b ions identifying the
six C-terminal amino acids of VTA2. The precision of this
experiment did not allow lysine to be distinguished from
glutamine.
Cell lines and cultures
Peripheral blood lymphocytes were obtained from hepari-
nized venous blood of healthy volunteers using Ficoll-Paque
(Amersham Pharmacia Biotech AB, Uppsala, Sweden).
Fresh NK cell populations were obtained by peripheral
blood lymphocyte depletion of non-NK cells using the NK
Cell Isolation Kit (Miltenyi Biotec GmbH, Bergisch-
Gladbach, Germany). The phenotype of isolated cells was

analyzed by flow cytometry with CD16 mAb and CD56
mAb (clone 3G8 and N901-NKH1, respectively; Immuno-
tech-Beckman-Coulter, Marseilles, France). The resulting
NK population typically comprised more than 85% of
CD56 cells and less than 0.5% CD3 cells. The different
human cell targets (K562, an MHC class I-deficient mono-
myelocytic tumor [28]; Daudi, a b2m
–
Burkitt’s lymphoma
cell line [29]; Val, a non-Hodgkin B cell lymphoma [30];
C1R, a lymphoblastoid tumor expressing only the HLA
class I allele Cw 0401 [31]; and C1R-B27 [32]) were
maintained in culture in complete RPMI 1640 medium
with Glutamax-I supplemented with 10% fetal calf serum
(Gibco–BRL, Life Technologies, Cergy Pontoise, France).
ThemurineFccR
+
mastocytoma P815 cell line was
maintained in complete Dulbecco’s modified Eagle’s culture
medium (Sigma Aldrich, St Louis, MO, USA) supplemen-
ted with 5% human serum. The human interleukin-2-
dependent NK cell lines NKL [33] and NK-92 [34] (kindly
provided by E. Vivier, CIML, Marseilles, France) were
maintained in RPMI 1640 medium with Glutamax-I
(Gibco–BRL) supplemented, respectively, with 10% pooled
human AB serum plus recombinant interleukin-2
(100 UÆmL
)1
; Chiron) and 10% fetal calf serum plus
recombinant interleukin-2 (200 UÆmL

)1
). The cd cell line
has been described previously [27].
All the culture media were complemented with
100 UÆmL
)1
penicillin, 100 UÆmL
)1
streptomycin and
1m
M
sodium pyruvate (all from Gibco–BRL).
Bioactivity assays
The cytolytic activity was assessed in a 4-h
51
Cr-release assay
in which effector cells (NKL, NK-92, freshly isolated NK or
cd cytotoxic T lymphocytes) were mixed with different
target cells. Lysis assays were carried out with or without Va
fractions added at different concentrations. Briefly, 10
6
target cells were labeled with 100 lCi sodium [
51
Cr]bichro-
mate (10 mCiÆmL
)1
;ICN)at37°C for 1 h. The cells were
then washed three times with RPMI and added to the
effector cells at 3 · 10
3

cells/well in 96-well round-bottom
microplates, resulting in an effector to target cell (E/T) ratio
ranging from 30 : 1 to 3 : 1 in a final volume of 0.2 mL in
each well. For the redirected killing assay, target cells were
2592 J. Tabiasco et al.(Eur. J. Biochem. 269) Ó FEBS 2002
the (FccR
+
) P815 cell line, and effector cells the CD16
+
NKL cell line, using an E/T ratio of 3 : 1. The assays were
carried out with or without 2 lgÆmL
)1
anti-CD16 IgG1
(clone 3G8). After 4 h of incubation at 37 °C, 100 lL
supernatant was harvested and counted in the gamma
counter. The percentage specific
51
Cr release was deter-
mined from the relation:
[(experimental c.p.m. ) spontaneous c.p.m.)/(total c.p.m.
incorporated ) spontaneous c.p.m.)] · 100
All determinations were performed in triplicate in each
assay, and results shown are the mean ± SEM from
triplicate determinations of a representative experiment out
of five independent experiments (on average). For clarity,
SEMs are not represented on most of the figures because
they were below 3% of specific lysis.
Release of tumor necrosis factor a (TNF-a)wasmeas-
uredbyabioassayusingTNF-a-sensitive cells (WEHI-
13VAR, ATCC CRL-2148). Briefly, 5 · 10

4
NKL cells per
well were incubated for 24 h at 37 °C with or without
various concentrations of each viscotoxin in 100 lLculture
medium. A 50-lL portion of supernatant was then added to
50 lL WEHI cells plated at 3 · 10
4
cells per well in culture
medium containing actinomycin D (2 lgÆmL
)1
)andLiCl
(40 m
M
). WEHI cells were incubated for 20 h at 37 °C.
Viability of WEHI cells was then measured with a 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(Sigma) assay. Levels of TNF-a releasewerethencalculated
from a standard curve obtained using purified human
recombinant TNF-a (PeproTech, Inc., Rocky Hill, NJ,
USA). Typically, unstimulated NKL cells release
5±2ngÆmL
)1
TNF-a compared with 60 ± 5 ngÆmL
)1
when induced by phorbol myristate acetate (4 ngÆmL
)1
)and
ionomycin (500 ngÆmL
)1
).

To probe for soluble cytolytic factors released by NK
cells during the lysis assay, 9 · 10
4
NK cells were mixed
with 3000 K562 cells and incubated with viscotoxins for 4 h
at 37 °C. A 100-lL portion of supernatant was then added
to 3000
51
Cr-labeled K562 cells plated in a 96-well round-
bottom microplate. After 4 h incubation at 37 °C, 100 lL
supernatant was collected and counted in a gamma counter.
The percentage of specific
51
Cr release was determined as for
the cytotoxicity assay.
RESULTS
Nontoxic concentrations of Va extract enhance NK
cell-mediated and antibody-redirected lysis
A crude hydrosoluble extract was prepared from crushed
mistletoe leaves, and added to in vitro lysis assays with K562
tumor targets and various NK effector cells. As indicated by
the spontaneous release of
51
Cr from target cells alone, Va
extract diluted 1 : 5000 was not directly toxic for these
targets. On the other hand, Va extract increased K562 lysis
by freshly isolated NK cells or by a strongly cytolytic cd T
cell line [35]. NK cell lysis in the presence of Va extract was
Fig. 1. V. album extract (VaE) enhances
cytolytic activity of different killer cells for

various target cells. (A) Lysis of K562 target
cells by the following effector cells: fresh NK
cells, cd cytotoxic T lymphocytes, NKL, and
NK-92 titrated at different E/T ratios, in the
presence (d) or absence (s)ofVaextract
diluted 1 : 5000 in all assays. Shown on the
right of each lysis assay are the c.p.m. of
spontaneous
51
Cr release by the target cells
alone (target spontaneous release) incubated
under the same conditions in the presence
(filled bars) or absence (empty bars) of Va
extract diluted 1 : 5000 in all assays. Each
result represents the mean of triplicate experi-
ments. (B) NKL effectors were tested for kill-
ing of the target cells lines, C1R, C1R-B27,
Daudi and Val, at different E/T ratios, in the
presence (d) or absence (s)ofVaextract
diluted 1 : 5000. Shown on the left of each
cytotoxic experiment are the c.p.m. of spon-
taneous
51
Cr release by the target cells alone as
above. (C) NKL cells were tested in a redi-
recting killing assay against the FccR
+
P815
target cells. These assays were carried out in
the absence or presence of CD16 mAb

(2 lgÆmL
)1
) and with or without Va extract
(VaE) diluted 1 : 5000. Each result represents
the mean of triplicate experiments at an E/T
ratio of 3 : 1.
Ó FEBS 2002 Viscotoxins enhance NK cell-mediated killing (Eur. J. Biochem. 269) 2593
equivalent to four times the number of killer cells when
present alone (Fig. 1A). Similar results were found with two
other NK cell lines, NK-92 and NKL. Thus nontoxic
concentrations of Va extract increased NK lysis.
NK cells eliminate tumor cells in the absence of
appropriate interactions between their inhibitory receptors
and MHC class I molecules on the surface of the target cells.
Thus we investigated whether Va extract would alter the
lysis of target cells other than K562 by the cell line NKL. Va
extract also enhanced killing of the HLA class I-deficient
targets C1R and Daudi (Fig. 1B). However, Va extract did
not trigger killing of the HLA class I cells Val or C1R-B27, a
C1R cell line transfected with the HLA-B27 allele protecting
against lysis by NKL cells [33].
NK cells may also lyse IgG-bound tumor cells [36], so we
investigated whether Va extract was also bioactive in this
case. Lysis of the FccRIII
+
cells P815 by NKL cells in the
presence of CD16 mAb [37] was measured with and without
Va extract. Again, Va extract was not directly toxic to P815
cells but enhanced its antibody-redirected killing (Fig. 1C).
Therefore, the bioactivity of this Va extract appears to be

independent of the NK-lysis activation pathway.
Bioactivity-guided isolation of thermostable
and low-molecular-mass proteins from Va extract
Bioactivity of Va extract in NKL cells could be titrated in
lysis assays by varying the E/T ratio and Va extract
concentrations (Fig. 2A). This helped to guide the isolation
of the molecule(s) responsible. After various treatments of
Va extract, titration of the remaining bioactivity indicated
that the bioactive component(s) were resistant to heating,
acid, protease, phosphatase and sodium periodate (Ta-
ble 1). This suggested that the molecule was thermostable,
although presumably neither a protein nor a carbohydrate,
despite their relative abundance (carbohydrate content
10 mgÆmL
)1
; data not shown) in Va extract revealed by
silver nitrate and Coomassie Blue staining of SDS/poly-
acrylamide gels (Fig. 2B). However, bioactivity in the Va
extract was degraded by alkali or a reduction–alkylation
treatment that targeted disulfide bridges (Table 1). Further
isolation of the NK-bioactive compound(s) was based on
HPLC with macroporous reverse-phase support (Fig. 2C).
Bioassay-guided fractionation of the Va extract indicated
that the bioactivity was due to distinct UV-absorbing peaks
split into two consecutive HPLC fractions. The HPLC
fractions 14–17 min and 17–20 min increased the NKL-
mediated lysis of K562 cells (Fig. 2C). With regard to the
specific NK lysis, no other bioactive fraction could be
recovered by this procedure. Both absorbance characteris-
tics of the active fractions (not shown) and their appearance

on SDS/PAGE (Fig. 2B) revealed that they comprised
small protein(s) with estimated molecular mass of 5 kDa.
The only Va extract molecules bioactive in NK lysis
are VTA1-3
On the one hand, proteins were unexpected for the unusual
thermostability and protease-insensitivity of the bioactive
extract, but, on the other hand, they could account for the
Fig. 2. NK cell lysis bioassay-guided isolation
of viscotoxins. (A) Cytolytic activity of NKL
cells against K562 target cells with Va extract
diluted 1 : 5000 (j), 1 : 15 000 (m), 1 : 45 000
(e) or without Va extract (s). (B) SDS/
PAGE of Va extract and fraction 17–20 min
(VT)stainedwitheitherAgNO
3
or Coomassie
Blue. (C) RP-HPLC separation of bioactive
fractions from the Va extract monitored for
total ion current and for bioactivity on NKL
cells. Fractions were collected every 3 min,
lyophilized, and tested in the NKL/K562
killing assay as above. Online coupling of
HPLC to ion trap electrospray ionization MS
was used to identify viscotoxins at the specified
peaks.
2594 J. Tabiasco et al.(Eur. J. Biochem. 269) Ó FEBS 2002
sensitivity to disulfide-specific reaction (Table 1). To unam-
biguously identify these molecules, we used online coupling
of the above HPLC separation with ion-trap MS detection
(liquid chromatography/MS). Using positive-mode electro-

spray, analysis of the HPLC peaks gave a series of
multicharged ion species (not shown) that were further
deconvoluted in the 2000–6000 mass range. The deconvo-
lution spectrum of the major peak from bioactive fraction
17–20 min gave two molecular species of 4827.0 and
4925.0 Da (Fig. 3A). They correspond, respectively, to the
expected molecular mass of VTA2 (theoretical mass 4828.4)
and to a noncovalent phosphate adduct of VTA2 [25].
Similar analysis of the other bioactive HPLC peaks
identified VTA1 (mass 4884.0) and VTA3 (mass 4830.0)
(Fig. 2C and [25]). This conclusion was checked by partial
peptide sequencing using MS
n
. As the N-terminal moiety of
native viscotoxins involves a disulfide bridge [25], sequential
fragmentation of each C-terminal moiety was performed to
confirm these assignments. From native VTA2, we selected
byMS
2
two ions belonging to the Y series (m/z 407, 706.3)
(data not shown). Its fragmentation into the expected
Y-type and b-type fragments characterized the C-terminal
PSDYPK hexapeptide sequence (Fig. 3B). Similar experi-
ments established the identity of VTA1 and VTA3 (data not
shown).
The HPLC fraction eluted at 17–20 min was dried and
weighed, and, although its bioactivity in NK-mediated
killing was clear-cut, it still comprised a mixture of distinct
viscotoxins. The same reverse-phase HPLC procedure was
repeated, but using a slower elution sequence. This resulted

in resolution into three peaks corresponding to each of the
three distinct viscotoxins. Each of the separated viscotoxins
was then individually controlled chromatographically by
reverse-phase (Fig. 4A) and cation-exchange (Fig. 4B)
chromatography. Parallel bioactivity monitoring of these
runs revealed that NK lysis activity was recovered with each
of the purified viscotoxin peaks. A molecular mass of
4884 Da identified VTA1 in the first bioactive fraction (not
shown), a molecular mass of 4827 Da identified VTA2 in
the second bioactive fraction (Fig. 4A), and a molecular
mass of 4830 Da (and 4928 Da for the phosphate adduct)
identified VTA3 in the third bioactive fraction (Fig. 4A).
The bioactivities of VTA1–3 were destroyed by alkali and
reduction–alkylation of disulfide bridges, but not by heat or
periodate, as described above for Va extract (Table 1). So,
this profile establishes the involvement of VTA1–3 as the
only components of Va extract that enhance NK lysis.
Moreover, the fact that polymyxin B (10 lgÆmL
)1
) did not
affect the bioactivity of the purified viscotoxins ruled out
any contribution of traces of lipopolysaccharide to the
observed effect (not shown).
Table 1. NK bioactivity of Va extract and purified viscotoxins after
chemical and enzymatic treatments. The NK lysis-increasing bioactivity
was normalized to 100% using the largest increase in lytic response in
the presence of untreated Va extract (here + 22% of specific K562
lysis), VTA1 (here + 16%) or VTA2 (here + 20%). For comparison,
the NK lysis-increasing bioactivity of chemically treated extracts is
expressed as a percentage of the reference bioactivity. Va extract,

VTA1 and VTA2 were used at 1 : 5000 dilution, 100 n
M
and 40 n
M
,
respectively, which had no direct toxicity for K562 cells. NT, Not
tested; DTT, dithiothreitol; IEt, iodoethanol.
Treatment
NK bioactivity (%)
Va extract VTA1 VTA2
None 100 100 100
Heat (75 °C, 2 h) 91 74 88
Dilution in organic solvent
(80% acetonitrile)
73 100 100
Acid
(2
M
HCl, 2 h, 37 °C)
67 NT NT
Alkali
(2
M
NaOH, 2 h, 37 °C)
13 0 0
Periodate oxidation
(5 m
M
NaIO
4

, 2 h, 20 °C)
100 91 120
Reduction + alkylation
(DTT, IEt)
000
Proteinase K 90 NT NT
Alkaline phosphatase 77 NT NT
Sulfatase 100 NT NT
Fig. 3. Biochemical identification of the visco-
toxin A2. (A) Deconvoluted spectrum from
the electrospray ionization MS analysis of the
VTA2 peak corresponds to the expected mass
of VTA2. The amino-acid sequence with
disulfide bridges of VTA2 is shown on the
right [25]. (B) MS
3
spectrum of the ion at m/z
706.3 generated from pentacharged VTA2
(m/z 966.6). The sequence of the C-terminal
moiety and assignment of the fragmentation
series are indicated.
Ó FEBS 2002 Viscotoxins enhance NK cell-mediated killing (Eur. J. Biochem. 269) 2595
Using SDS/PAGE examination of serial Va extract
dilutions, we estimated the total viscotoxin concentration in
the Va extract to be  3m
M
. This estimate is in concor-
dance with the respective bioactivities measured above for
purified viscotoxin and Va extract.
Each purified viscotoxin species was quantified by

analytical HPLC, and its bioactivity in the NKL/K562
assay titrated by serial dilutions. As each of the viscotoxin
samples produced similar increases in NK lysis, we
compared the three viscotoxins by determining their
respective EC
50
values, i.e. the VTA concentration produ-
cing half-maximal increase in NK-mediated lysis. These
EC
50
values are significantly different: 85 ± 6.4 n
M
for
VTA1, 18 ± 4.2 n
M
forVTA2and8±3.0n
M
for VTA3
(two-by-two comparison, highest two-tailed P value for
statistical difference equalled 0.0284). These findings
matched that of an authentic VTA3 standard [24], which
enhanced NK-mediated lysis with EC
50
¼ 6.5 ± 3.5 n
M
without direct cytotoxicity within the 1–100 n
M
range
(Fig. 5). This reference VTA3 EC
50

value was not signifi-
cantly different from that of the VTA3 (P ¼ 0.6031)
purified in this study. Together these data confirm that
viscotoxins are the Va extract compounds responsible for
the increase in NK cell-mediated cytotoxicity.
VTA2 acts on NK–target cell conjugates
The interaction between NK and target cells successively
involves the formation of conjugates, the specific recogni-
tion phase, and the lethal hit delivery. Afterwards, the killer
cells are recycled and may serially kill several targets [38].
Because the action of viscotoxin was found to be independ-
ent of the NK activation pathway (Fig. 1), we investigated
Fig. 5. Effect of a VTA3 standard on NK-mediated lysis in the absence
of direct cytotoxicity. Lysis of K562 target cells by NK cells at E/T ratio
of 1 : 1 in the absence (0) or presence of pure VTA3 standard (kindly
provided by K. Urech [24]) added in the concentration range 0–100 n
M
to the culture wells (up). Toxicity of VTA3 for the target cells alone
was titrated over the same concentration range as above by measure-
ment of the direct
51
Cr release from pulsed K562 cells (down). Each
result represents the mean of triplicate experiments.
Fig. 4. Biochemical characterization of the
purified viscotoxins A1, A2 and A3. (A)
RP-HPLC and (B) cation-exchange HPLC of
purified viscotoxins A2 and A3 monitored for
absorbance at 220 nm and for bioactivity on
NKL cells. Fractions delimited by tick marks
were collected, lyophilized, and tested in the

NKL/K562 killing assay. The deconvoluted
mass spectrum of each purified viscotoxin is
shownintheinset.
2596 J. Tabiasco et al.(Eur. J. Biochem. 269) Ó FEBS 2002
whether a viscotoxin pulse of either NKL effector or K562
target cells before the assay could also lead to this
bioactivity. Neither VTA2-pulsed killers nor VTA2-pulsed
targets alone reproduced the bioactivity of viscotoxins in the
usual lytic assay (Fig. 6A). Therefore, to exert its bioactiv-
ity, viscotoxin must be present during the killing assay.
Moreover, viscotoxins did not induce production of soluble
toxic factors by NKL cells during the 4-h incubation with
target cells (data not shown; see Materials and methods).
These results indicate that VTA2 acts on NK–target cell
conjugates.
From cell mixing to final cell harvest, these cytotoxicity
assays span 4 h, so we determined viscotoxin bioactivity
when added at various time points. The optimum for VTA2
bioactivity was when it was added  30–45 min after cell
contact (Fig. 6B). When compared with other drugs
targeting NK activation [39], this time-course suggests that
VTA2 affected an event subsequent to conjugate formation,
in agreement with the former conclusion.
VTA2 does not activate NK cells
NK–target cell conjugation precedes NK cell activation,
which follows a complex array of activatory and inhibitory
receptor–ligand interactions. In hematopoietic cells, intra-
cellular transduction of activation signals involves changes
in metabolic rates, which may be detected by micro-
physiometry [27]. As Va extract acts on both redirected and

natural lysis by NK cells (Fig. 1), its effects appear to be
independent of the NK activation pathway. Therefore,
viscotoxins were not expected to directly activate NK cells.
To address this issue, we tested microphysiometric responses
of NKL cells to VTA2. Although a short pulse with the
mitogen combination of phorbol ester and ionomycin
increased the metabolic rate of NKL cells (Fig. 6C), no
such change was detected in parallel assays with NK cells
exposed to various concentrations of VTA2 alone (shown
for 50 n
M
in Fig. 6C) or to phorbol and VTA2 together
(data not shown). So VTA2 does not constitute an activator
signal to NK cells nor bring them a cosignal additional to
phorbol esters. This conclusion was also supported by the
finding that NKL cells incubated with VTA2 alone did not
produce any more TNF-a than unstimulated ones (data not
shown).
After killer–target cell binding and NK cell activation,
early intracellular events involved in NK-mediated lysis
comprise polarization and delivery of cytotoxic granules
at the target cell synapse. We investigated whether the
Fig. 6. VTA2 acts on NK–target cell conjugates without a change in extracellular acidification rate of NK cells and killing of bystander cells. (A)
Enhancement of NK cytotoxicity cannot be reproduced by a VTA2 pulse of effector or target cells before the lysis assay. The effector cells or target
cells were separately preincubated with 50 n
M
VTA2 for 4 h and washed before running the usual cytotoxic assay without VTA2. For comparison,
the same assay was run without preincubation and with or without VTA2 during the killing assay. (B) Time course of VTA2 bioactivity in the killing
assay. At t ¼ 0 min, effector and target cells were mixed. At the times indicated, 50 n
M

VTA2 (final concentration) was added, and 4 h after
mixing of the cells, specific
51
Cr release was determined. For each time point, viscotoxin bioactivity was calculated as follows: (% of specific lysis
with VT) ) (% of specific lysis without VTA2). For each time point, the viscotoxin bioactivity was normalized using the largest increase found with
viscotoxin in the assay (+15% at t ¼ 30 min; s). For comparison, the time course of bioactivity of ion channel blockers was plotted (d)as
described by Sidell et al. [39]. (C) NKL cells in microphysiometer chambers were drained with complete medium only (n), with complete medium
containing phorbol myristate acetate + ionomycin (m) or with complete medium containing 50 n
M
VTA2 (s). For 80 min, extracellular
acidification was monitored every 90 s in each chamber, and the response was normalized to the rate value before sample introduction. (D)
Bioactivity of VTA2 was tested in a three-cell lysis assay involving NKL killer, K562 target and C1R-B27 bystander cells. While the presence of
bystander cells did not interfere with viscotoxin bioactivity in the killing assay (left), VTA2 did not induce any detectable killing of
51
Cr-labeled
bystander cells (right).
Ó FEBS 2002 Viscotoxins enhance NK cell-mediated killing (Eur. J. Biochem. 269) 2597
killing-enhancing bioactivity of VTA2 resulted from altered
polarization of lytic granules in such a way as to kill
bystander cells in addition to bound targets. NKL cells were
simultaneously exposed to K562 target cells and C1R-B27
bystander cells (one target for one bystander mixed
together). We then measured the effect of VTA2 on either
lysis of
51
Cr-labeled K562 target cells in the presence of
bystanders, or reciprocally, on lysis of
51
Cr-labeled C1R-
B27 bystanders in the presence of K562 target cells. VTA2

was devoid of direct toxicity to the bystander cells while
simultaneously being bioactive against the NK targets
(Fig. 6D).
So, whereas VTA2 enhances killing of K562 target cells in
the presence of bystanders, it does not induce further
collateral lysis and hence does not affect the polarization of
the cytolytic activity of NK cells.
DISCUSSION
The lytic activity of human NK cells is enhanced by
V. album compounds [10,40,41]. Previous studies have
investigated this effect using fermented Va extracts,
peripheral blood lymphocytes, and cytokine-dependent
and lymphokine-dependent contexts [13,15,42]. The
results presented here indicate that the lysis of K562
cells by either freshly isolated NK cells or the tumoral
NK cell lines NK-92 and NKL is increased by the
presence of Va extract molecules in the lytic assay. The
lytic potential of killer cells in the presence of the extract
was almost the same as that of four times as many killer
cells alone. This effect of Va extract is not restricted to
NK cells, as other types of cytotoxic T lymphocyte, such
as cd T cells, that exert strong NK-like activity against
thesametargetsarealsoenhancedbythesemolecules.
So we investigated the V. album-mediated enhancement
of killing by NK cells independently of exogenous
cytokine supply.
In previous studies, the V. album molecules bioactive in
tumor cell killing by NK cells and monocyte/macrophages
were identified as oligosaccharides (arabinogalactan [11] or
rhamnogalacturonan [12,43]), which link killer and target

cells [43]. This report describes the isolation and identifica-
tion of VTA1, VTA2 and VTA3 as the only Va extract
component of NK-cell-mediated lysis. Mistletoe produces
two protein families with related biological properties:
lectins and viscotoxins [44,45]. Viscotoxins are a group of
highly basic cysteine-rich small proteins related to the family
of thionins [46] and notoriously resistant to protein-
denaturing agents [45,47]. Table 1 shows that the starting
bioactivity of the Va extract has such characteristics. Other
workers had already reported an uncharacterized Va-
derived peptide that activated NK cells in vivo [48]. Thionins
are cytotoxic for a large variety of eukaryote and proka-
ryote cells [49]. This toxicity relies on positive charges of
thionins interacting unspecifically with phospholipids. Thus,
by passive insertion into the cell membranes, thionins
permeabilize and kill tumoral cells [49,50]. Few studies have
in fact elucidated the basis of viscotoxin toxicity. Using the
most viscotoxin-sensitive cell line [51], micromolar concen-
trations of viscotoxins are toxic for the rat renal sarcoma
Yoshida [24]. More recently, VTA3 has been found to
induce cell membrane stiffening and destabilization [52].
Here, the action on NK-cell-mediated killing of tumor
targets was analysed using nontoxic nanomolar concentra-
tions of viscotoxin. The data presented lead to clearer
conclusions than earlier studies of the effect of V. album on
tumor cell lysis by monocytes and NK cells. The synergistic
enhancement of lysis by mistletoe arabinogalactan is
mediated by cytokines secreted from killer cells after its
binding to NK surface receptors [13,15]. These cells pulsed
with high concentrations of arabinogalactans killed more

target cells [13,15].
Our study shows a different spectrum of effects of
purified viscotoxins which appear to be focused on estab-
lished killer–target cell conjugates.
NK cell lysis results from intracellular integration of a
wide array of receptor-mediated activatory and inhibitory
signals. As the absence of a lytic response could result from
dominance of negative signaling by cell targets, the actual
activity of viscotoxins on positive signaling pathways
needed to be investigated in NK cells more directly.
Activation of hematopoietic cells involves increased glyco-
lysis, Na
+
/H
+
antiport, and lactate and carbonic acid
excretion, which together cause medium acidification [53].
Microphysiometry monitors extracellular acidification as a
real-time follow-up of metabolic rate, and detected NKL
response to a mitogen combination. VTA2 alone does not
alter the metabolic rate of NKL cells, so they do not provide
a self-sufficient positive signal for activating NK cells.
Furthermore, the action of VTA2 on killer–target cell
couples spares the ÔunproductiveÕ killer-bystander ones.
Therefore VTA2 does not alter the polarization of cytolytic
activity against the activating target, nor does it weaken the
latter in such a way as to increase their lysis in the assay.
Besides the lack of viscotoxin toxicity for the various targets
cells in this study, this conclusion can be drawn from the
several lines of evidence discussed below. First, a pulse and

rinse of targets with viscotoxins did not increase their
subsequent lysis by NK cells. Secondly, viscotoxins do not
cause the lysis of bystander cells exposed to viscotoxins and
activated NK.
Investigation of the molecular basis of viscotoxin action
on NK–target cell conjugates was not within the scope of
this study. However, the high bioactivity of viscotoxins (i.e.
in the nanomolar range) in this assay, and the differential
(8–80 n
M
) of bioactivity between the structurally related
VTA1–3 proteins strongly suggest an interaction with an
unidentified receptor involved in NK lysis. Viscotoxins
are structurally related to thionins [46], which behave
as selective ion channels in nanomolar concentration
ranges [50]. As functional ion channels are involved in
NK-mediated lysis [54], a related role for viscotoxins is
hypothesized.
Long-standing medicinal virtues have been imputed to
mistletoe extracts, used in the treatment of some solid
tumors. Although mistletoe was thought to owe much of
its bioactivity to the direct toxicity of several components
to tumoral cells, this report shows that highly purified
viscotoxins alone enhance the efficiency of tumor cell
lysis by NK cells in the absence of any direct cytotoxicity
to the target. Although the mode of viscotoxin action
has not been defined, we show that viscotoxins are
involved in immunologically mediated tumour cell
destruction. Future studies will aim to identify the
viscotoxin receptors involved in this therapeutically

interesting bioactivity.
2598 J. Tabiasco et al.(Eur. J. Biochem. 269) Ó FEBS 2002
ACKNOWLEDGEMENTS
We wish to thank E. Vivier for helpful discussions and provision of cell
lines, and E. Espinosa for the cd cell line. We also thank Dr K. Urech
for the gift of the highly purified viscotoxin A3. This work was
supported by institutional grants from INSERM and Programme
Eureka from ARC.
REFERENCES
1. Leroi, R. (1977) Nachbehandlung des operierten MammaKar-
zinoms mit Viscum album. Helv. Chim. Acta 44, 403–414.
2. Friess, H., Beger, H.G., Kunz, J., Funk, N., Schilling, M. &
Buchler, M.W. (1996) Treatment of advanced pancreatic
cancer with mistletoe: results of a pilot trial. Anticancer Res. 16,
915–920.
3. Heiny, B.M., Albrecht, V. & Beuth, J. (1998) Correlation of
immune cell activities and beta-endorphin release in breast carci-
noma patients treated with galactose-specific lectin standardized
mistletoe extract. Anticancer Res. 18, 583–586.
4. Zarkovic,N.,Vukovic,T.,Loncaric,I.,Miletic,M.,Zarkovic,K.,
Borovic,S.,Cipak,A.,Sabolovic,S.,Konitzer,M.&Mang,S.
(2001) An overview on anticancer activities of the Viscum album
extract Isorel. Cancer Biother. Radiopharmacol. 16, 55–62.
5. Steuer-Vogt, M.K., Bonkowsky, V., Ambrosch, P., Scholz, M.,
Neiss, A., Strutz, J., Hennig, M., Lenarz, T. & Arnold, W. (2001)
The effect of an adjuvant mistletoe treatment programme in
resected head and neck cancer patients: a randomised controlled
clinical trial. Eur. J. Cancer 37, 23–31.
6. Gabius, S. & Gabius, H.J. (1999) [Immunomodulating mistletoe
therapy by lectin standardization: a double-edged sword? (see

comments) ]. Versicherungsmedizin 51, 128–136.
7. Gabius, H.J., Andre, S., Kaltner, H., Siebert, H.C., von der
Lieth, C.W. & Gabius, S. (1996) The mistletoe myth: claims,
reality and provable perspectives. Z. Arztl. Fortbild. (Jena) 90,
103–110.
8. Baxevanis, C.N., Voutsas, I.F., Soler, M.H., Gritzapis, A.D.,
Tsitsilonis, O.E., Stoeva, S., Voelter, W., Arsenis, P. &
Papamichail, M. (1998) Mistletoe lectin I-induced effects on
human cytotoxic lymphocytes. I. Synergism with IL-2 in the
induction of enhanced LAK cytotoxicity. Immunopharmacol.
Immunotoxicol. 20, 355–372.
9. Valentiner, U., Pfuller, U., Baum, C. & Schumacher, U. (2002)
The cytotoxic effect of mistletoe lectins I, II and III on sensitive
and multidrug resistant human colon cancer cell lines in vitro.
Toxicology 171, 187–199.
10. Hamprecht,K.,Handgretinger,R.,Voetsch,W.&Anderer,F.A.
(1987) Mediation of human NK-activity by components in
extracts of Viscum album. Int. J. Immunopharmacol. 9, 199–209.
11. Wagner, H. & Jordan, E. (1988) An immunologically active ara-
binogalactan from Viscum album ÔberriesÕ. Phytochemistry 27,
2511–2517.
12. Mueller, E.A., Hamprecht, K. & Anderer, F.A. (1989) Biochemical
characterization of a component in extracts of Viscum album
enhancing human NK cytotoxicity. Immunopharmacology 17,
11–18.
13. Mueller, E.A. & Anderer, F.A. (1990) A Viscum album oligo-
saccharide activating human natural cytotoxicity is an interferon
gamma inducer. Cancer Immunol. Immunother. 32, 221–227.
14. Mueller, E.A. & Anderer, F.A. (1990) Synergistic action of a plant
rhamnogalacturonan enhancing antitumor cytotoxicity of human

natural killer and lymphokine-activated killer cells: chemical spe-
cificityoftargetcellrecognition.Cancer Res. 50, 3646–3651
(erratum appears in Cancer Res. 50, 6115).
15. Hauer, J. & Anderer, F.A. (1993) Mechanism of stimulation of
human natural killer cytotoxicity by arabinogalactan from Larix
occidentalis. Cancer Immunol. Immunother. 36, 237–244.
16. Fischer, S., Scheffler, A. & Kabelitz, D. (1996) Activation of
human gamma delta T-cells by heat-treated mistletoe plant
extracts. Immunol. Lett. 52, 69–72.
17. Fischer, S., Scheffler, A. & Kabelitz, D. (1997) Stimulation of the
specific immune system by mistletoe extracts. Anticancer Drugs 8
(Suppl. 1), S33–S37.
18. Trinchieri, G. (1989) Biology of natural killer cells. Adv. Immunol.
47, 187–376.
19. Colonna, M., Moretta, A., Vely, F. & Vivier, E. (2000) A high-
resolution view of NK-cell receptors: structure and function.
Immunol. Today 21, 428–431.
20. Tomasello, E., Blery, M., Vely, E. & Vivier, E. (2000) Signaling
pathways engaged by NK cell receptors: double concerto for
activating receptors, inhibitory receptors and NK cells. Semin.
Immunol. 12, 139–147.
21. Moretta, A., Biassoni, R., Bottino, C., Mingari, M.C. &
Moretta, L. (2000) Natural cytotoxicity receptors that trigger
human NK-cell-mediated cytolysis. Immunol. Today 21, 228–234.
22. Moretta, L., Biassoni, R., Bottino, C., Mingari, M.C. & Moretta,
A. (2000) Human NK-cell receptors. Immunol. Today 21, 420–422.
23. Lanier, L.L. (2001) On guard-activating NK receptors. Nat.
Immmunol. 2, 23–27.
24. Schaller, G., Urech, K. & Giannattasio, M. (1996) Cytotoxicity of
different viscotoxins and extracts from the European subspecies of

Viscum album L. Phytother. Res. 10, 473–477.
25. Orru,S.,Scaloni,A.,Giannattasio,M.,Urech,K.,Pucci,P.&
Schaller, G. (1997) Amino acid sequence, S-S bridge arrangement
and distribution in plant tissues of thionins from Viscum album.
Biol. Chem. 378, 989–996.
26. Nitti,G.,Orru,S.,Bloch,C.,Morhy,L.,Marino,G.&Pucci,P.
(1995) Amino acid sequence and disulphide-bridge pattern of three
gamma-thionins from Sorghum bicolor. Eur. J. Biochem. 228,
250–256.
27. Espinosa, E., Belmant, C., Pont, F., Luciani, B., Poupot, R.,
Romagne
´
, F., Brailly, H., Bonneville, M. & Fournie
´
, J.J. (2001)
Chemical synthesis and biological activity of bromohydrin pyr-
ophosphate a potent phosphoantigen stimulating human cd T
cells. J. Biol. Chem. 276, 18337–18344.
28. Jondal, M. & Pross, H. (1975) Surface markers on human B and T
lymphocytes. VI. Cytotoxicity against cell lines as a functional
marker for lymphocyte subpopulations. Int. J. Cancer 15,
596–605.
29. Klein, E., Klein, G., Nadkarni, J.S., Nadkarni, J.J., Wigzell, H. &
Clifford, P. (1968) Surface IgM-kappa specificity on a Burkitt
lymphoma cell in vivo and in derived culture lines. Cancer Res. 28,
1300–1310.
30. Deweindt, C., Kerckaert, J.P., Tilly, H., Quief, S., Nguyen, V.C. &
Bastard, C. (1993) Cloning of a breakpoint cluster region at band
3q27 involved in human non-Hodgkin’s lymphoma. Genes Chro-
mosomes Cancer 8, 149–154.

31. Litwin, V., Gumperz, J., Parham, P., Phillips, J.H. & Lanier, L.L.
(1993) Specificity of HLA class I antigen recognition by human
NK clones: evidence for clonal heterogeneity, protection by self
and non-self alleles, and influence of the target cell type. J. Exp.
Med. 178, 1321–1336.
32. DeMars, R., Rudersdorf, R.,Chang, C., Petersen, J., Strandtmann,
J., Korn, N., Sidwell, B. & Orr, H.T. (1985) Mutations that impair
a posttranscriptional step in expression of HLA-A and -B anti-
gens. Proc. Natl Acad. Sci. USA 82, 8183–8187.
33. Robertson, M.J., Cochran, K.J., Cameron, C., Le, J.M.,
Tantravahi, R. & Ritz, J. (1996) Characterization of a cell line,
NKL, derived from an aggressive human natural killer cell
leukemia. Exp. Hematol. 24, 406–415.
34. Gong, J.H., Maki, G. & Klingemann, H.G. (1994) Characteriza-
tion of a human cell line (NK-92) with phenotypical and func-
tional characteristics of activated natural killer cells. Leukemia 8,
652–658.
Ó FEBS 2002 Viscotoxins enhance NK cell-mediated killing (Eur. J. Biochem. 269) 2599
35. Ferrarini, M., Heltai, S., Toninelli, E., Sabbadini, M.G., Pellicciari,
C. & Manfredi, A.A. (1995) Daudi lymphoma killing triggers the
programmed death of cytotoxic V gamma 9/V delta 2 T
lymphocytes. J. Immunol. 154, 3704–3712.
36. Perussia, B. (2000) Signaling for cytotoxicity. Nat. Immunol. 1,
372–374.
37. Perussia, B., Trinchieri, G. & Cerottini, J.C. (1979) Functional
studies of Fc receptor-bearing human lymphocytes: effect of
treatment with proteolytic enzymes. J. Immunol. 123, 681–687.
38. Kupfer, A. & Singer, S.J. (1989) Cell biology of cytotoxic and
helper T cell functions: immunofluorescence microscopic studies
of single cells and cell couples. Annu. Rev. Immunol. 7, 309–337.

39. Sidell, N., Schlichter, L.C., Wright, S.C., Hagiwara, S. & Golub,
S.H. (1986) Potassium channels in human NK cells are involved in
discrete stages of the killing process. J. Immunol. 137, 1650–1658.
40. Hajto, T. (1986) Immunomodulatory effects of iscador: a Viscum
album preparation. Oncology 43, 51–65.
41. Schink, M. (1997) Mistletoe therapy for human cancer: the role of
the natural killer cells. Anticancer Drugs 8 (Suppl. 1), S47–S51.
42. Stein, G.M. & Berg, P.A. (1997) Mistletoe extract-induced effects
on immunocompetent cells: in vitro studies. Anticancer Drugs 8
(Suppl. 1), S39–S42.
43. Mueller, E.A. & Anderer, F.A. (1990) Chemical specificity of
effector cell/tumor cell bridging by a Viscum album rhamno-
galacturonan enhancing cytotoxicity of human NK cells.
Immunopharmacology 19, 69–77.
44. Jung, M.L., Baudino, S., Ribereau-Gayon, G. & Beck, J.P. (1990)
Characterization of cytotoxic proteins from mistletoe (Viscum
album L.). Cancer Lett. 51, 103–108.
45. Park, J.H., Hyun, C.K. & Shin, H.K. (1999) Cytotoxic effects of
the components in heat-treated mistletoe (Viscum album). Cancer
Lett. 139, 207–213.
46. Florack, D.E. & Stiekema, W.J. (1994) Thionins: properties,
possible biological roles and mechanisms of action. Plant Mol.
Biol. 26, 25–37.
47. Bruneton, J. (1999) Pharmacognosie.3rdedn.EditionsMe
´
dicales
Internationales, Paris.
48. Kuttan, G. & Kuttan, R. (1992) Immunomodulatory activity of a
peptide isolated from Viscum album extract (NSC 635, 089).
Immunol. Invest. 21, 285–296.

49. Carrasco, L., Vazquez, D., Hernandez-Lucas, C., Carbonero, P. &
Garcia-Olmedo, F. (1981) Thionins: plant peptides that modify
membrane permeability in cultured mammalian cells. Eur.
J. Biochem. 116, 185–189.
50. Hughes, P., Dennis, E., Whitecross, M., Llewellyn, D. &
Gage, P. (2000) The cytotoxic plant protein, beta-purothionin,
forms ion channels in lipid membranes. J. Biol. Chem. 275,
823–827.
51.Urech,K.,Schaller,G.,Ziska,P.&Giannasttasio,M.(1995)
Comparative study on the cytotoxic effect of viscotoxin and
mistletoe lectin on tumor cells in culture. Phytother. Res. 9,
49–55.
52. Coulon, A., Berkane, E., Sautereau, A.M., Urech, K., Rouge, P. &
Lopez, A. (2002) Modes of membrane interaction of a natural
cysteine-rich peptide: viscotoxin A3. Biochim. Biophys. Acta 1559,
145–159.
53. McConnell, H.M., Owicki, J.C., Parce, J.W., Miller, D.L., Baxter,
G.T., Wada, H.G. & Pitchford, S. (1992) The cytosensor micro-
physiometer: biological applications of silicon technology. Science
257, 1906–1912.
54. Schlichter, L., Sidell, N. & Hagiwara, S. (1986) Potassium chan-
nels mediate killing by human natural killer cells. Proc. Natl. Acad.
Sci. USA 83, 451–455.
2600 J. Tabiasco et al.(Eur. J. Biochem. 269) Ó FEBS 2002