invalid, the S phase (of an individual egg)
could be as short as the time needed for the
doubling of the largest replicating unit (ap-
prox. 4min, Fig. I c). Our finding that 14%
of the molecules are in replication is fully
compatible with both of these extreme pos-
sibilities.
We thank Mrs. A. Poppitz and Mrs. I.
Schotz for valuable technical assistence.
Supported by DFG Mo 158/14.
Received May 12, 1980
1. Blumenthal, A.B., et al.: Cold Spring Har-
bor Syrup. Quant. Biol.
38,
205 (1973)
2. DNA: Replication and Recombination.
Cold Spring Harbor Syrup. Quant. Biol.
43
(1979)
3. Stinchcomb, D.T., et al.: Nature
282, 39
(1979)
4. Baldari, C.T., et al.: Cell 15, 1095 (1978)
5. Scheinin, R., et aI. : Ann. Rev. Biochem.
47,
277 (1978)
6. Nemer, M. : J. Biol. Chem.
237,
143 (1962)
Cell Fusion Induced by High Electric
to
Dictyostelium
E. Neumann, G. Gerisch and K. Opatz
Max-Planck-Institut ffir Biochemie, D-8033 Martinsried
When cells of the eukaryotic microorgan-
ism
Dictyostelium discoideum
are repeat-
edly exposed to electric fields, they fuse
into multinuclear, motile cells, the largest
ones containing more than 40 nuclei. Un-
der optimal conditions, i.e., initial field in-
tensities of 4 to 6 kV/cm with decay times
of about 40 ps, lysis of cells as a result
of membrane permeabilization is negligi-
ble. This means that cells can be fused
extensively without substantial loss.
The apparatus used for generating high elec-
tric impulses of short duration has been
described elsewhere [1]. Most of our exper-
iments were carried out using the
D. dis-
coideum
strain Ax-2, which is adapted to
growth in liquid medium [2]. However, sim-
ilar results were obtained with a wild-
type strain, v-12/M2, grown in suspension
on
E. coil B/r
as a nutrient.
Impulses of the initial field intensity Eo
decaying exponentially with time accord-
ing to
E(t) = Eo"
exp (-
t/RC)
were applied
(R resistance of the cell suspension ; C ca-
pacitance of the discharge circuit). Cells
were suspended in 17 mM Soerensen phos-
phate buffer pH 6.0 at a density of 107
or 2.10 s cells/ml and, in the latter case,
bubbled with oxygen before transferring
1-ml samples into a chamber equipped
with two flat platinum electrodes separated
by a distance d- 1.4 cm. At the lower den-
sity the cells had to be agglutinated by
rolling the suspensions in plastic tubes in
order to produce maximal contact between
the cells before subjecting them to field
impulses. Agglutination strongly increased
the yield of fused ceils.
Repeated impulses given at intervals of 1
to 3 s were more efficient than single ones.
Impulses Applied
Our standard procedure consisted of 3 or
4 impulses applied at intervals of 3 s. Celt
fusion was found to have occurred as soon
as the cells could be removed from the
electrode chamber and put under a micro-
scope, i.e., within the first few seconds after
the repeated exposure to an electric field.
MgC1 z or CaC12 at a concentration of
2 mM inhibited fusion when present dur-
ing field application. Since the absence of
divalent cations resulted in delayed lysis
of the cells as manifested at about 0.5 h
after field application, the fused cells were
diluted into phosphate buffer containing
2 mM CaC12.
When cells of strain Ax-2 were used, best
results were obtained with impulses of an
initial field intensity Eo = 5.7 kV/cm. With
a R-- 1.8.103 s and C=21 nF, the decay
time constant was
RC=
38 ps. The temper-
ature increase due to Joule heating was
calculated as A T-~ 0.18 ~ per pulse; hence
the temperature increase appears to be too
small for facilitating cell fusion. Quantita-
tive data for a typical experiment are given
in Fig. 1.
65
60
55
50
45
4O
35
control
cells
cells exposed to
electric fields
~25
20
15
IO
5
0
1 5 I0 15 20 25 33 35 40
45
nuclei per cell
Fig. 1. Histogram showing the increased number of nuclei residing after electric field application
in multinuclear cells. 35% of all nuclei were found in cells containing )10 nuclei, and 9%
in cells containing )30 nuclei. Cells of these sizes were absent from the controls. In the
experiment shown, cells of
D. diseoideum
strain Ax-2 were grown in nutrient medium as described
previously [7] and harvested during exponential growth. Cells were washed in 17 mM Soerensen
phosphate buffer pH 6.0 and resuspended in the buffer at a density of 107 cells/ml. Fifteen
minutes before field application the cells were allowed to agglutinate under gentle shaking
in a roller tube. An electric field of Eo = 5.7 kV/cm was applied three times at intervals of
3 s at 23 ~ Samples of 100 or 200 btl were transferred onto the Teflon membrane of Petriperm
dishes (Heraeus, Hanau, Germany) in order to supply the ceils with oxygen. Five rain thereafter
3 ml of 2 mM CaClz solution in phosphate buffer was added, and the cells were allowed
to settle and spread on the Teflon surface. The cells were fixed on the surface in 70% ethanol.
Nuclei were counted after staining with Mithramycin, a fluorescent stain for DNA [8] which
brilliantly labeled nuclei and mitochondria
414 Naturwissenschaften 67 (1980) 9 by Springer-Verlag 1980
Field intensities of 7 kV/cm or higher
caused lysis of many of the cells. Likewise,
with a higher capacitance C=52 nF and
a resulting decay time constant of RC=
104 ~ts, a large fi'action of the cells was
lost because of substantial lysis, even when
the initial field intensity was kept at
E0=5.7 kV/cm, Wild-type cells of the v-
12/M2 strain were more sensitive than
those of Ax-2. Consequently, the field in-
tensity was reduced for v- 12/M2 to 4.3 kV/
cm and the number of impulses limited
to 2 in order to avoid substantial lysis.
Electric fields may cause drastic permeabil-
ity changes in the membranes of various
cells and organelles [1, 3]. The primary ef-
fect of the field impulses is a transient and
local perturbation of the structure of the
membrane, which only slowly anneals after
pulse termination [4]. In agglutinated cells
of D. discoideum the plasma membranes
are in contact with each other. Field-
induced perturbations at sites of adhesion
are apparently the starting points for the
formation of larger cells by membrane fu-
sion.
Cells of D. discoideum appear to contain
no sialic acid at their surfaces, hence they
are devoid of receptors for Sendal virus,
one of the best fusing agents of mamma-
lian cells [5]. Also, polyethylene glycol,
now the most widely used fusing agent [6],
did not, in our hands, produce cell fusion
in D. discoideum. Thus, electric impulses
provide the method of choice for cell fu-
sion in this organism. The electric field
method might be adaptable to fusion of
other cells also.
In D. discoideum the method opens a possi-
bility for mass production of heteroka-
ryons. These can be used in genetic studies
as a source of recombinants, as well as
in biochemical investigations on the com-
plementation between different mutant nu-
clei residing in a common cytoplasm. In
this context it is of importance that giant
multinuclear cells produced shortly after
the end of growth are able to develop and
to participate in aggregation.
We thank C.-R. Rabl for cooperation. Our
work was supported by the Deutsche For-
schungsgemeinschaft.
Received April 21, 1980
1. Neumann, E., Rosenheck, K.: J. Membr.
Biol. 10, 279 (1972)
2. Watts, D.J., Ashworth, J.M.: Biochem. J.
119, 171 (1970)
3. Sale, A.J.H., Hamilton, W.A.: Biochim.
Biophys. Acta 163, 37 (I 968); Zimmermann,
U., Schulz, J., Pilwat, G.: Biophys. J. 13,
1005 (1973); Kinosita, K., Tsong, T.Y.:
Proc. Nat. Acad. Sci. USA 74, 1923 (1977)
4. Rosenheck, K., Lindner, P., Pecht, I.: J.
Membr. Biol. 12, 1 (1974); Harbich, W.,
Helfrich, W.: Z. Naturforsch. 34a, 1063
(1979); Lindner, P., Neumann, E., Rosen-
beck, K.: J. Membr. Biol. 32, 231 (1977)
5. Poste, G.: Int. Rev. Cytol. 33, 157 (1972);
Poste, G., Nicolson, G.L. (eds.): Cell Sur-
face Reviews, Vol. 5. Amsterdam: Norttr-
Holland 1978
6. Kao, K.N., Michayluk, M.R.: Planta 155,
355 (1974); Pontecorvo, G., Riddle, P.N.,
Hales, A.:Nature 265, 257 (.1977)
7. Malchow, D., et al.: Eur. J. Biochem. 28,
136
(1972)
8. Crissman, H.A., Tobey, R.A.: Science 184,
1297 (1974)
Tumor Inhibition by Titanocene Dichloride:
First Clues to the Mechanism of Action
P. K6pf-Maier
Institut fiir Anatomie der Freien Universitfit, D-1000 Berlin-Dahlem
H. K6pf
Institut ffir Anorganische Chemie der Technischen Universitfit,
D-1000 Berlin-Charlottenburg
Metallocene dichlorides, (CsHs)zMCla, ex-
hibit cancerostatic activity against the Ehr-
lich ascites tumor (EAT') system in mice.
Optimum activity leading to 100% cure
rates is observed with M=Ti [1-3], V [4],
Nb [5], and Mo [6]. The mechanism of ac-
tion of this species of organometallic com-
plexes is yet unknown.
Recently the directly cytostatic and cyto-
toxic effects ofmetallocene dichlorides have
been demonstrated in vitro by determina-
tion of the cell growth inhibition and by
vital staining [7]. In the present study we
report on the influence of an in vivo treat-
ment with titanocene dichloride on the fol-
lowing in vitro incorporation of 3H-la-
belled, specific precursors of the DNA,
RNA, or protein synthesis, respectively,
into the acid-insoluble fraction of EAT
cells.
About 6.106 EAT cells per animal were
transplanted intraperitoneally (i.p.) on
60 female CF1 mice. Four days later the
tumor was well developed. Now 30 ani-
mals received a single i.p. injection of
90 mg titanocene dichloride/kg, prepared
and purified according to [8], and dissolved
in 0.4 ml of a mixture of dimethylsulfoxide
and saline (v/v= 1/9). Another 30 animals
served as untreated controls and obtained
an i.p. injection of 0.4 ml of only the di-
methylsulfoxide-saline mixture. At various
intervals up to 96 h after injection (Fig.
1) cells were removed in equal amounts
(determination by use of an electronic par-
ticle counter Coulter Counter DN) each
from 2 treated and 2 untreated animals
and equally distributed to 3 samples per
animal. Each sample contained 5 ml mini-
E o,
80 i DNA
40 ~ _ 0 0 ............ ,~.....0
24 48 72 96 [h]
80 ~ RNA
i
,7
.......... EL---O
40 tio_
oo ....
o o
0 .if"
0 ~'L~'"L%'~
24 48 72 96
[It]
I~] i.
L J~, Protein • z~
40
IA,~.o ./'" ZX
0 ~ -- , ,
24 48 72 96 [h~
Fig. 1. Relative incorporation rates of [methyl-
3H]thymidine, [5-3H]uridine, and L-
[4,5-3H]leucine as measures of the DNA,
RNA, and protein syntheses in EAT cells. At
0 h in vivo treatment with 90 mg titanocene
dichloride/kg. Cell harvesting after the resi-
dence times in vivo given on the abscissa, fol-
lowed by an incorporation period of 20 min
in vitro
Naturwissenschaften 67 (1980) 9 by Springer-Verlag 1980 415