AbstractThe cytolytic action of cardiotoxin analogue III from the venom of the Formosan cobra on chick embryonal fibroblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus was investigated. The 50% effective dose of the toxin for the cells cultured at a non-permissive temperature (41°C) or for noninfected normal cells was about 8 μgml whereas the value was 2 μgml for the cells cultured at a permissive temperature (36°C). This indicates that the transformed cells became more susceptible to the cytolytic action of the toxin than the non-transformed cells

HIDETO KANESHIMA

KIYOHIDE KOJIMA

KYOZO HAYASHI

MICHINARI HAMAGUCHI

NORIO KANEDA

KANEDA, NORIO

26166

HAMAGUCHI, MICHINARI

26458

KOJIMA, KIYOHIDE

26459

KANESHIMA, HIDETO

26460

HAYASHI, KYOZO

24106

Gif University: Repository / 岐阜薬大学術リポジトリ

Action of Cobra Venom Cardiotoxin on Chick Embryonal Fibroblasts Transformed with a Temperature-Sensitive Mutant of Rous Sarcoma Virus.

Kaneda, Norio

Hamaguchi, Michinari

Kojima, Kiyohide

Kaneshima, Hideto

Hayashi, Kyozo

Elsevier - Publisher Connector 

Action of cobra venom cardiotoxin on chick embryonal fibroblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus 

Volume 192, number 2 FEBS 3139 November 1985 
Action of cobra venom cardiotoxin on chick embryonal 
fibroblasts transformed with a temperature-sensitive mutant 
of Rous sarcoma virus 
Norio Kaneda, Michinari Hamaguchi*, Kiyohide Kojima*, Hideto Kaneshima” and 
Kyozo Hayash? 
Department of Biochemistry, *Research Institute for Disease Mechanism and Control, “Department of Pathology, Nagoya 
University School of Medicine, Nagoya 466 and +Department of Biology, Gtjii Pharmaceutical University, Gijii 502, Japan 
Received 26 August 1985; revised version received 9 October 1985 
The cytolytic action of cardiotoxin analogue III from the venom of the Formosan cobra on chick embryonal 
fibroblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus was investigated. The
50% effective dose of the toxin for the cells cultured at a non-permissive temperature (41°C) or for non- 
infected normal cells was about 8 pg/ml whereas the value was 2 pg/ml for the cells cultured at a permissive 
temperature (36°C). This indicates that the transformed cells became more susceptible to the cytolytic action 
of the toxin than the non-transformed cells. 
Cobra venom cardiotoxin Cytotoxicity Rous sarcoma virus Temperature-sensitive mutant Fibroblast 
Membrane lipidfluidity 
1. INTRODUCTION 
Cobra venom cytotoxins (cardiotoxins) are 
highly basic membrane-active polypeptides con- 
sisting of 60-61 amino acid residues with 4 in- 
tramolecular disulfide linkages [l-3]. They exhibit 
in vitro a weak direct hemolytic effect and relative- 
ly preferential cytotoxicity toward tumor cells such 
as Yoshida sarcoma and other ascites hepatoma 
cells [4,5]. In previous studies, comparison of the 
cytolytic activity toward normal and transformed 
cells was carried out for different species or among 
different cell species [6]. Therefore, it was difficult 
to appreciate the difference in susceptibility be- 
tween transformed and non-transformed cells. 
Abbreviations: t.s, temperature-sensitive; CTX-III, car- 
diotoxin analogue III; RSV; Rous sarcoma virus; DPH, 
l,ddiphenyl-1,3,5-hexatriene; FCS, fetal calf serum; 
PBS, Dulbecco’s phosphate-buffered saline 
’ To whom correspondence should be addressed 
Here, we describe the cytolytic action of the major 
toxin, CTX-III (cardiotoxin of Narita and Lee [7]), 
on chick embryonal fibroblasts infected with a ts 
mutant of RSV with which a strict control experi- 
ment can be performed with the same cell species 
by only changing the temperature. 
2. MATERIALS AND METHODS 
2.1. Materials 
CTX-III was purified from the venom of the 
Formosan cobra (Naja naja atra) as described [8]. 
It was found to be homogeneous by a high- 
performance liquid chromatography [9]. A ts mu- 
tant of RSV, strain LA-3 1, isolated by Wyke [lo], 
belonging to the class T mutant (a defect in 
transformation) which can transform the cells at a 
permissive temperature (36”(Z), but not at a non- 
permissive temperature (41”C), was kindly sup- 
plied by Dr M. Yoshida (Cancer Institute, Tokyo). 
Published by Elsevier Science Publishers B. V. (Biomedical Division) 
00145793/85/$3.30 0 1985 Federation f European Biochemical Societies 313 
Volume 192, number 2 FEBS LETTERS November 1985 
2.2. Culture of fibroblasts 
Chick embryonal fibroblasts were prepared 
from 1 l-day embryos by trypsinization and cul- 
tured in medium 199 supplemented with 10% FCS 
and 10% tryptose phosphate broth as described 
[ll]. Subconfluent cells were infected with the 
virus at 36°C for 15 h in the presence of polybrene 
(lO,ug/ml), and cultured at 36°C for 2 weeks until 
all cells were morphologically transformed. Since 
long-term incubation at 36°C resulted in a decrease 
of cell viability and appearance of vacuoles in 
cytoplasm, transformed cells were incubated at 
41 “C for 2 days and transferred to 36°C for an ad- 
ditional 2 days before use. After mild treatment 
with trypsin (500 pg/ml, 20-30 s at room tempe- 
rature), cells were suspended by careful pipetting 
in PBS containing 1 mM EDTA, and then washed 
twice with PBS containing 2% FCS and adjusted 
to approx. 2 x lo6 cells/ml. 
2.3. Cytotoxicity assay 
A mixture of 50~1 each of a cell suspension and 
a serial dilution of CTX-III in PBS containing 2% 
FCS was incubated for 30 min at 37°C. The reac- 
tion was terminated by cooling the mixture in an 
ice bath, and then a 50~1 aliquot of 0.4% trypan 
blue in PBS was added for the dye exclusion test 
[8]. The number of intact and damaged cells were 
determined on a hemocytometer slide glass. 
2.4. Fluorescence labeling 
Cells were harvested in the same manner as 
described above until the first centrifugation step. 
The subsequent washing buffer was changed to 
PBS containing 5 mM MgCL and 1 mM CaC12. 
These divalent cations were effective for maintain- 
ing cell viability during fluorescence labeling. 
After washing with this buffer the cell suspension 
was adjusted to approx. 0.8 x lo6 cells/ml. A DPH 
dispersion in PBS (2 PM) was prepared according 
to [12]. Labeling was carried out for 1 h at 25°C 
as in [12] in the presence of 5 mM MgCl2 and 1 mM 
CaCl2. After washing with PBS containing 2% 
FCS, the cells were resuspended in the same buffer 
for the fluorescence measurement. The viability of 
the labeled cells was over 90%. 
2.5. Fluorescence polarization measurements 
Fluorescence polarization was measured with an 
Elscint model MV-la microviscosimeter (Elscint, 
314 
Israel). The degree of fluorescence polarization, P, 
was directly recorded. The rotational relaxation 
time (Q) of the fluorophore was calculated with the 
following Perrin equation; 
r0 
-=1+’ 
r e 
where r-0 and r are the limited and measured 
fluorescence anisotropy, respectively, and 7 is the 
fluorescence lifetime of the excited state of DPH 
[13]. The r-0 value was 0.362 [13] and r was calcu- 
lated from P [14]. 
3. RESULTS AND DISCUSSION 
Chick embryonal fibroblasts infected with the ts 
mutant of RSV were reversibly transformed on 
shifting down of the temperature from a non- 
permissive (41°C) to a permissive temperature 
(36°C). Non-transformed cells had a flat and 
smooth cell surface as non-infected cells, and 
transformed cells showed a round form with 
numerous microvilli which was evident after 2 days 
incubation at 36°C (fig. 1). These morphological 
features were the same as those described [15]. The 
dose-response curves for the cytolytic activity of 
CTX-III toward these cells are shown in fig.2. 
With the infected cells cultured at 41”C, the effec- 
tive dose for 50070 lysis of the cells (EDSO) was 
about 8 pg/ml, which was almost identical to that 
for normal fibroblasts without viral infection. 
With the cells cultured at 36°C the EDso value 
decreased significantly to 2 /rg/ml. At this concen- 
tration, the non-transformed cells retained over 
90% viability. These changes in susceptibility 
toward the toxin were reversible, depending on the 
cultivation temperature. These facts may suggest 
that the increased susceptibility to CTX-III is one 
of the transformational phenotypes of the cells. 
The membrane lipid fluidity of 3 kinds of cells 
was measured by the fluorescence depolarization 
method using DPH, and the relative fluidity was 
expressed in terms of the rotational relaxation time 
(Q) of the fluorophore (table 1). It was found that 
non-transformed cells gave similar Q values to 
those for normal fibroblasts, whereas transformed 
cells resulted in rather lower values at each tempe- 
rature measured. This suggests that the membrane 
lipid fluidity increases upon transformation. 
Volume 192, number 2 FEBSLETTERS November 1985 
Fig. 1. Scanning electron micrographs of chick embryonal fibroblasts infected with a ts mutant of Rous sarcoma virus, 
strain LA-31. Cells cultured at (A) 41°C for 2 days, (B) at 36°C for 2 days. Bar, 5 pm. 
0 1 5 10 50 100 
CARDIOTOXIN-ANALOGUE Ill (vg/mll 
Fig.2. Dose-response curves of the cytolytic action of 
CTX-III on chick embryonal fibroblasts infected with 
the ts mutant of Rous sarcoma virus. The cell suspension 
(2 x lo6 cells/ml) and toxin were incubated for 30 min 
at 37°C in PBS containing 2910 FCS. Cytotoxicity was 
assayed by the dye exclusion test. Cells cultured at a (0) 
non-permissive temperature (41”C), (0) permissive 
temperature (36’C); (A) fibroblasts which were not in- 
fected with the virus. Data represent means f SE for 3 
independent experiments. The statistical differences bet- 
ween non-transformed (41°C) and transformed (36°C) 
cells were calculated according to Student’s t-test; 
* P<O.O5; ** P<O.Ol. 
Table 1 
Degree of fluorescence polarization (P) and rotational 
relaxation time (Q) of DPH embedded in the plasma 
membrane of chick embryonal fibroblasts infected with 
the ts mutant of Rous sarcoma virus 
Tempera- Transformed Non-trans- Normal 
ture (“C) cells formed cells fibroblasts 
P e (ns) P e W P e (ns) 
4 0.292 49 0.305 55 0.306 56 
20 0.248 30 0.269 36 0.261 33 
37 0.192 15 0.213 18 0.213 18 
P was measured at 3 different temperatures for cells 
cultured at 36°C (transformed) and 41°C (non- 
transformed), and normal fibroblasts which were not 
infected with the virus. e was calculated as described in 
the text assuming the fluorescence lifetime of DPH to be 
11.0, 10.5 and 8.8 ns at 4,20 and 37°C respectively [13]. 
The data represent he mean values for 2 independent 
experiments 
Recently, it was proposed that the interpretation of 
membrane lipid fluidity should be done with care 
when intact cells are investigated because hydro- 
carbon fluorophores such as DPH label the intra- 
cellular membrane region as well as the plasma 
315 
Volume 192, number 2 FEBS LETTERS November 1985 
membrane. Grunberger et al. [16] described a 
method for distinguishing plasma membrane lipid 
fluidity from intracellular fluidity, and reported 
that the plasma membrane had almost the same 
lipid fluidity as intracellular membranes in the 
cases of 3T3 fibroblasts and macrophages. This 
method might be useful for further investigations 
to confirm our present results. It would be of in- 
terest to consider that the membrane lipid fluidity 
plays an important role in the cytolytic action of 
CTX-III and that cell transformation induces a 
change in lipid fluidity of the plasma membrane 
which may lead to modification of the susceptibili- 
ty of the cells to the toxin. 
ACKNOWLEDGEMENT 
We thank Dr S. Saga (Nagoya University School 
of Medicine) for carrying out the scanning electron 
microscopy. 
REFERENCES 
ill 
PI 
[31 
[41 
PI 
WI 
171 
PI 
191 
DOI 
1111 
1121 
v31 
1141 
[151 
[I61 
Lee, C.Y. (1972) Ann. Rev. Pharmacol. 12, 
265-286. 
Yang, C.C. (1974) Toxicon 12, l-43. 
Narita, K., Cheng, K.L., Chang, W.C. and Lo, 
T.B. (1978) Int. J. Peptide Protein Res. 11, 
229-237. 
Braganca, B.M., Patel, T.N. and Badrinath, P.G. 
(1967) Biochim. Biophys. Acta 136, 508-520. 
Patel, T.N., Braganca, B.M. and Bellare, R.A. 
(1969) Exp. Cell Res. 57, 289-297. 
Iwaguchi, T., Takechi, M. and Hayashi, K. (1985) 
Biochem. Int. 10, 343-349. 
Narita, K. and Lee, C.Y. (1970) Biochem. Biophys. 
Res. Commun. 41, 339-343. 
Kaneda, N., Sasaki, T. and Hayashi, K. (1977) Bio- 
chim. Biophys. Acta 491, 53-66. 
Kaneda, N. and Hayashi, K. (1983) J. Chromatogr. 
281, 389-392. 
Wyke, J.A. (1973) Virology 52, 587-590. 
Saga, S., Hamaguchi, M., Hoshino, M. and 
Kojima, K. (1985) Exp. Cell Res. 156, 45-56. 
Shinitzky, M. and Inbar, M. (1974) J. Mol. Biol. 
85, 603-615. 
Shinitzky, M. and Barenholz, Y. (1974) J. Biol. 
Chem. 249, 2652-2657. 
Kaneda, N., Tanaka, F. and Yagi, K. (1983) J. 
Biochem. 94, 1317-1328. 
Wang, E. and Goldberg, A.R. (1976) Proc. Natl. 
Acad. Sci. USA 73, 4065-4069. 
Grunberger, D., Haimovitz, R. and Shinitzky, M. 
(1982) Biochim. Biophys. Acta 688, 764-774. 
316 


https://gifu-pu.repo.nii.ac.jp/?action=repository_action_common_download&item_id=8081&item_no=1&attribute_id=18&file_no=1

Action of Cobra Venom Cardiotoxin on Chick Embryonal Fibroblasts Transformed with a Temperature-Sensitive Mutant of Rous Sarcoma Virus.

Abstract

Similar works

Full text

Available Versions

Gif University: Repository / 岐阜薬大学術リポジトリ

Elsevier - Publisher Connector

Elsevier - Publisher Connector