J. Cell Sci. 9) 71-84(1971)
71
Printed in Great Britain
THE TOXIC EFFECT OF SPERMIDINE ON
NORMAL AND TRANSFORMED CELLS
H. OTSUKA
Medical Research Council Virology Unit, Institute of Virology,
University of Glasgow, Glasgow, Scotland
SUMMARY
The cytotoxic effect of spermidine was investigated on various tissue culture cell lines in the
presence of calf serum. Cells incubated with cytotoxic concentrations remained rounded up
while cells in control cultures always spread out on the surface of the Petri dish.
The spermidine concentration tolerated depends on cell number, concentration of serum,
the strain of cells used and the phase of the growth cycle.
The spermidine index (SI) of a cell culture is defined as the highest level of spermidine which
did not show cytotoxic effect in the standard test system. The SI measures the ability of a cell
culture (or line) to neutralize the cytotoxic effect of spermidine.
The SI of normal fibroblastic cells such as BHK21IC13 or mouse embryo cells alters characteristically with different phases of the cell cycle. It is highest in lag or early exponential phase,
then it diminishes during the exponential growth phase reaching its lowest point after the cell
culture has become confluent.
It is characteristic of polyoma virus-transformed cell lines and of other established permanent
cell lines (which are probably of spontaneously transformed origin) that their SI decreases only
slightly at high cell densities. There is a correlation between higher SI of transformed cells and
their ability to grow in soft agar suspension.
INTRODUCTION
Biological polyamines such as spermidine and spermine are widely distributed in
bacteria and fungi as well as in mammalian cells (Herbst & Snell, 1949; Rosenthal &
Tabor, 1956; Herbst, Weaver & Keister, 1957). It has been reported that these polyamines have a potent cytotoxic effect on tissue culture cells in the presence of calf
serum, while no such effect is observed with horse or human serum (Alarcon, Foley &
Modest, 1961; Alarcon, 1964; Bachrach, Abzug & Bekierkunst, 1967). The interpretation has been that polyamines are not cytotoxic by themselves but become so by the
action of monoamine oxidase which is known to be present in calf serum but not in
human or horse serum (Werle & Roewer, 1952; Hirsch, 1953; Tabor, Tabor &
Rosenthal, 1954). The cytotoxic agents are probably oxidatively deaminated products
of polyamines which have active aldehyde groups (Alarcon, 1964; Tabor, Tabor &
Bachrach, 1964).
This communication describes a simple method for assaying the cytotoxic effect
of oxidized spermidine on tissue culture cells. This assay method is then used for
comparative tests on various tissue culture cell lines and on cells in various stages of
the cell culture's growth cycle and the general conclusions that can be drawn are
discussed.
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MATERIALS AND METHODS
Establis/ied cell lines
The Syrian hamster cell line BHK21JC13 (Macpherson& Stoker, 1962; Macpherson, 1963)
and the recloned polyoma virus-transformed derivatives PyY (Stoker, 1962) were most commonly used. Another derivative, BHK21/C 13/rif 75, resistant to rifampicin up to 75/£g/ml
(Subak-Sharpe et al. 1970) was made available by Prof. Subak-Sharpe and mouse cell lines C3,
C10, polyoma virus-transformed rat cell line (RPTi), and adenovirus type 2 transformed rat
cell line (i?Ti) were kindly supplied by DrJ. F.Williams and Mr S. Ustacelebi in this laboratory.
Mouse L, HeLa, Hep-2 and BSC-i were also used and were freshly prepared.
Preparation of fresh clones of polyoma-transformed BHK21JC13
lines
6
A monolayer of BHK21JC13 cells (1 x io cells/50-mm Petri dish) was infected with 02 ml
of polyoma virus suspension (around io8 pfu) for 2 hat 37 °C. Then the cells were washed with
Eagle's medium, trypsinized and the monodisperse cells transferred into soft agar suspension
cultures as described by Macpherson & Montagnier (1964). After 10 days incubation 11 single
polyoma-transformed colonies growing in the agar suspension were isolated by Pasteur pipette.
They were propagated by passaging twice a week and all showed the growth characteristics of
transformed cells.
Virus
Using a small plaque variant derived from the Toronto strain of polyoma virus, a virus stock
was prepared from secondary mouse embryo cultures by treating the infected cell debris with
receptor-destroying enzymes (Crawford, 1962). The stock titre was 5 x io8 pfu/ml.
Medium
All cell lines were propagated at 37 °C in 20-oz (570-ml) flat bottles or 80-oz (2-3-I.)
Winchester bottles using modified Eagle's medium (Eagle, 1959) supplemented with 10%
tryptose phosphate broth and 10% unheated calf serum (10% ETC).
Standard growth cultures
Confluent monolayers from the 20- or 80-oz bottles were trypsinized, washed twice with 10 %
ETC and finally suspended in the same medium at a concentration of io6 cells/ml; 50-mm
plastic Petri dishes were seeded with 5 ml of this cell suspension and kept in a humidified CO2
incubator at 37 °C. After appropriate time intervals cells were trypsinized and suspended in
Eagle's medium supplemented with 10 % tryptose phosphate broth and 1 % unheated calf
serum (1 % ETC). An aliquot was taken for cell counting and the rest were subjected to the
standard spermidine test.
Standard spermidine test
The cell suspension prepared as above was centrifuged at low speed. The cell pellet was resuspended in 1 % ETC at io6 cells/ml, and distributed into 30-mm plastic Petri dishes in
2-ml aliquots. To each Petri dish 01 ml of spermidine trihydrochloride solution was added.
The concentrations of spermidine solution covered the range from 5 to 150 fig/ml in steps of
5 /ig/ml. The Petri dishes were incubated at 37 °C in a humidified COa incubator and after
15-20 h incubation cultures were examined with an inverted microscope. The cytotoxic effect
of spermidine was easily observable above a certain concentration of spermidine as shown in
Figs. 7-10. The maximum amount of spermidine which did not show cytotoxic effect in this
assay is defined as the spermidine index (SI).
Cytotoxic effect of oxidized spermidine
73
Trypsinization and cell counting
Unless otherwise specified trypsinization was carried out in the following way. After removing
the medium from the cell sheet, trypsin solution (0-25 % in tris-saline) was added then immediately removed. The cell sheet was left at room temperature until all cells had rounded off
from the Petri-dish surface, and then the cells were suspended in fresh medium. All cell lines
used gave single-cell suspensions by this method.
Cell suspensions were diluted to around io4 cells/ml with phosphate-buffered saline (Dulbecco & Vogt, 1954) and counted with a Coulter counter.
Analysis of radioactive spermidine treated cultures
Tests similar to those described above were carried out using [14C]spermidine except that
twice the standard number of cells were used. After the incubation the medium was removed
from each Petri dish with a Pasteur pipette, 2 ml of trypsin solution added, the cells therein
suspended and then separated from the trypsin solution by low-speed centrifugaiion. The cell
pellet was suspended in 2 ml of 5 % cold trichloroacetic acid (TCA) and the TCA-soluble
fraction separated by a further centrifugation. The TCA-insoluble fraction was collected on
Millipore filters (0-45 /im). The radioactivities of medium, trypsin-soluble and TCA-soluble
fractions were measured in a Nuclear Chicago liquid scintillation counter after preparing the
samples as follows: o-i ml of the solution was applied to a glass-fibre disk (Whatman GF/C,
21 mm), which was transferred after drying into vials containing toluene-based scintillation
fluid. The TCA-insoluble fractions collected on Millipore filters were treated and counted
like the glass-fibre disks.
Materials
Spermidine trihydrochloride and crystalline bovine serum albumin were purchased from
Sigma Chemical Co. and Spermidine-C-14-trihydrochloride (specific activity, 107 mCi/mM)
from The New England Nuclear Corporation.
RESULTS
Effect of spermidine on BHK21JC13
cells from confluent cultures
The cytotoxic effect of spermidine on BHK2IJC13 cells is shown in Figs. 7-10.
In the presence of 1 • 5 /tg of spermidine all cells are rounded (Fig. 10) and with 1 -o fig
some cells spread, but most remain rounded (Fig. 9). But at 0-5 fig spermidine, virtually all cells spread (Fig. 8). The cytotoxic effect is irreversible: once cells are
rounded by the action of spermidine, they never spread again even after extensive
washing with fresh medium, and no cell division is observed even with prolonged
further incubation. In this standard spermidine assay the maximum amount of
spermidine tolerated before an observable cytotoxic effect was 0-5 fig. This is a highly
repeatable characteristic. Hence the SI of confluent BHK2i/C 13 cells is 0-5/tg. As
Tabor et al. (1954) reported, spermidine did not show any cytotoxic effect in the
presence of horse or human serum which does not contain monoamine oxidase.
When the number of cells plated per dish was increased in the spermidine test
system, more spermidine was required for cytotoxic effect. In Fig. 1 the SI values are
plotted against the number of cells in the spermidine test system. This correlation
suggests that the cells possess some definite capacity to neutralize the cytotoxic effect
of spermidine. The possibility that spermidine being a strong base produced complexes with the cellular nucleic acids, thus avoiding oxidation by serum monoamine
74
H. Otsuka
oxidase, can be ruled out, because the experiments using labelled spermidine suggest
that very little radioactivity remains associated with the cells.
The distribution of radioactivity in cultures treated with labelled spermidine is
shown in Table i. No cytotoxic effect appeared in the culture treated with i"O/tg of
8
7
6
5
m 4
3
2
1
\ 4x10*
8x10= 12x10* 1-6x10*
Standard test .,
, ,,
, , ,
No. of cells per 2 ml culture
2x10»
Fig. i. The relation between SI and cell number. Spermidine tolerance tests with
different numbers of cells per culture all originating from confluent BHK21JC13
cell monolayers.
Table 1. Distribution of the radioactivity in the cultures
treated with [uC]spermidine
Concentration of [14C]spermidine...
2/tg
Total cpm
Medium
Trypsin-soluble fraction
TCA-soluble fraction
TCA-insoluble fraction
810
2160
40
%
Total cpm
96
159900
1
54°
03
3
1 960
40
1-2
98-5
labelled spermidine. In this culture 96% of the radioactivity was found in the medium,
while only 4% was associated with cells. Increasing the amount of spermidine to
2-0 /fg resulted in the unambiguous cytotoxic effect, but the distribution of the radioactivity was unchanged; 98% was present in the medium and 2% in the cells. The
radioactive compound in the medium was analysed by paper chromatography using
butanol:pyridine: acetic acid: water (15:10:3:12) as solvent. The RF value of spermidine in this system was 0-15. The major radioactive compound found in the medium
had an RF value of 0-24 and no spermidine was detected. The chemical structure of
Cytotoxic effect of oxidized spermidine
75
the compound is not identified but the above strongly suggests that the spermidine
was completely oxidized in both cultures. In a separate experiment it was found that
up to 20 fig of spermidine were completely oxidized in the standard spermidine test
system.
Another possibility is that the cells possess some capacity to neutralize the toxic
Table 2. Si-reducing activity of calf serum and of bovine serum albumin
Spermidine in culture, fig
2-0
Control
Serum (o'i ml)
Serum (0-2 ml)
Albumin (10 mg)
3'5
2-5
Amount
neutralized
corrected by
subtracting
4-0 control, fig
+
+
±
+
+
+
20
±
Serum and albumin solution (10 mg/o-i ml of Eagle's medium) were added to the standard
spermidine test system and the cytotoxic effect was examined. — , no effect; ± , partial effect;
+, complete effect.
10 -
<
so
60
70
80
90
Fractions
100
110
120
Fig. 2. Fractionation of calf serum proteins on Sephadex G-200 in relation to SIreducing activity. Twenty millilitres of calf serum were applied to a Sephadex G-200
column (25 x 900 mm) previously washed with O-OIM phosphate buffer (pH 7-0)
containing 015 M sodium chloride. The column was eluted with the same buffer and
2-9 ml fractions were collected. After measuring the optical density at 280 nm, 02 ml
of each fraction was pipetted into a 30-mm Petri dish, to which 1-5 fig of spermidine
and 2 ml of BHK21/C13 cell suspension (2 x io6 cells in 1 % ETC) were added. The
cultures were incubated at 37 °C for 16 h and examined for cytotoxic effect. Fractions
which neutralized the cytotoxic effect (reduced the SI) are indicated by crosshatching in the figure; where the effect was slight this is indicated by stippling.
76
H. Otsuka
substance produced by the oxidation of spermidine. To test this hypothesis the
following experiments were performed. Spermidine (15-0, 12-5, 100, 7-5, and 5-0/tg)
was incubated in a 30-mm Petri dish with 2 x io6 BHK21IC1J cells from a confluent
monolayer suspended in 2 ml of 1 % ETC. Controls without cells were also run. After
16 h incubation at 37 °C the cytotoxic effect was clearly seen in the cultures containing
more than 7-5 /tg of spermidine, while 7-5 and 5-0 /tg of spermidine failed to show any
cytotoxic effect. The medium was taken from each Petri dish and tested for persistence
of the cytotoxic effect against 2 x ioB fresh cells in the standard test system. If cells,
only while still alive, can neutralize the effect of spermidine, then it would be expected
that the medium containing more than io-o /ig of spermidine might retain cytotoxicity
while the rest would not. It was found that all the media from cell-containing cultures
had lost their cytotoxic effect. The controls which were incubated without BHK21IC IJ
cells showed that the cytotoxic substance produced by the oxidation of spermidine is
stable at 37 °C for at least 20 h. Hence 2 x io6 BHK21JC13 cells are able to neutralize
more than 15-0 /tg of spermidine, although they do not survive in the presence of
io-o /tg or more of spermidine.
Serum also neutralizes the cytotoxic effect of spermidine. This neutralizing or SIreducing activity of serum is retained in the dialysis bag, which indicates that it has
high molecular weight. Calf serum was fractionated by gel filtration on a Sephadex
G-200 column into 3 fractions as shown in Fig. 2. Si-reducing activity was only
detected in the last peak, which mostly consisted of albumin. The neutralizing activity
of purified serum albumin was therefore tested. In Table 2, Si-reducing activity of
calf serum and bovine serum albumin are compared; 0-2 ml of calf serum neutralizes
1-5 /tg of spermidine and 10 mg of albumin neutralize 2-0/tg. Since 0-2 ml serum contained about 8 mg of albumin (Schultze & Heremans, 1966), it can be concluded that
all the Si-reducing activity of serum is probably due to serum albumin. The molar
ratio between 10 mg albumin and 2-0/tg spermidine is 20:1, assuming the molecular
weight of albumin is 6-9 x io 4 and that of spermidine 255. As 20 molecules of albumin
appear to be required to neutralize one molecule of spermidine it appears unlikely that
the neutralizing effect is due to spermidine molecules being adsorbed to large albumin
molecules by their negative charges. If this were the case, many spermidine molecules
would be adsorbed on to one molecule of albumin.
Changes in SI of BHK21/C13 cells in various growth stages
All experiments reported so far were done with BHK21JC13 cells derived from
confluent cultures where the spermidine index is a reliable characteristic. However,
the SI varies greatly if the cells are obtained at different stages in the formation of
a monolayer.
BHK2IJC13 cells were seeded in Petri dishes as described in standard growth
cultures and harvested for testing at various stages up to the formation of a confluent
monolayer. The growth curve and SI are shown in Fig. 3. There is an initial lag of
more than 12 h, after which the cells number increases exponentially with a mean
doubling time of around 12 h. The SI at 5 h is 0-5, which is the same value as for confluent cells. It rises sharply at 12 h and reaches its maximum of 3-5 at 15 h. In the
Cytotoxic effect of oxidized spermidine
JJ
exponential phase it falls gradually and settles down to 0-5 when the monolayer has
reached confluence.
Higher SI of polyoma-transformed cell lines
Standard spermidine tests were carried out with various polyoma virus-transformed
BUK2I/C/J lines. The growth curve and SI of one such line (clone 7) are shown in
Fig. 4. The pattern of the SI changes during the growth stages in the formation of
8x10'
6x10'
4x10' -
4x10'
10
u 2x10'
o
o.
o
Z
10
2 2x10" -
o
Z
5x10'
5x10= -,
10
20
30
40
50
60
Incubation time of initial culture, h
Fig. 3
1 I
10 20 30 40 50 60
Incubation time of initial culture, h
Fig. 4
Fig. 3. The change in SI of BHK21JC13 cells in various growth phases. • , number of
cells per dish; O, SI.
Fig. 4. Change in SI of clone 7 of polyoma virus-transformed BHK21/C13 cells in
various growth phases. • , number of cells per dish; O, SI.
a monolayer as it does with BHK21JC13 cells, but the SI of transformed cells is much
higher at any growth stage: 4-fold higher at confluence and 2-fold higher when the
index is at its maximum. All 12 polyoma-transformed cell lines tested show such
higher SI values. The results are shown in Table 3. BHK21JC13 cells always gave
consistent results in repeated experiments, while the SI values of transformed cells,
both from the same clone and between clones, showed greater variation but they were
always well above that of BHK21JC13 cells.
The SI values of BHK21jC' 1j/rij'j•$ cells are also shown in Table 3. The morphology of this line is quite different from the original BHK21JC13 cells; it no longer
has a typical fibroblast appearance and grows in suspension of soft agar like polyomatransformed cells. The SI of this line is very high at confluence, 6 times higher than
that of BHK21JC13 cells, but the 15 h value is only 1-3 times that of BHK21IC13.
78
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Sublines of BHK21JC13 have been isolated which have many properties of transformed cells but have never been in contact with polyoma virus (Montagnier, Macpherson & Jarrett, 1966). They emerged spontaneously during prolonged passage of
Table 3. SI of various BHK21JC13 derived cell lines
Cell line
BHK21/C13
BHK2ijCr3lrif75
Polyoma-transformed
BHK21IC13
Clone 1
Clone 2
Clone 3
Clone 4
Clone s
Clone 6
Clone 7
Clone 8
Clone 9
Clone 10
Clone 11
PyY
SI at confluence
SI at 15 h
o-5
2-5, 3-o
30, 30, 35
45, 5-0
2-S. 2-5, 4-0
5-o, 7-5
5-o, 7-5
5-0, 7-5
5-o, 7-S
2-0, 3 0 ,
2-S. 3 0 , 3-0
2-5. 3-o, 3-o, 3'O, 3'5
2-0, 35
2-5, 2-5, 3'5, 4-o
2-0, 2-5, 4-5
2-O, 2-S, 2-S, 3-S
30
7-S. IS'©
7'S, 15-0
5-°, 7-5
5-o, 7-S
2-O
iS-o
15-0
io-o
3-o, 3 - o. 4'O
1 2 5 , 12-5, 1 5 0
2'O
The maximum SI was usually obtained using cells which had been incubated for 15 h under
standard growth conditions. The data represent all experimental results obtained except for
BHK21/C13 at confluence where a great many concordant experiments have been done.
Only one experiment was carried out on clones 9, 10 and 11.
ordinary BHK21IC13 cells. This does not necessarily rule out the possibility that
transformation was induced by unrecognized viruses which were present in the cells,
or through the cells becoming contaminated with unknown viruses during the
passages. It is therefore possible that the transformed-like morphology of BHK21I
Cij/rtf^ is not a consequence of adaptation to tolerate rifampicin. It is not known
at present whether spontaneously transformed cells have a selective advantage in the
presence of rifampicin.
SI of other cell lines
In the previous section it has been shown that polyoma-transformed or spontaneously
transformed BHKiijC 13 derived lines have higher SI than the original line. It is of
interest to test whether this is also true for other cell lines. Since most of the laboratoryestablished permanent cell lines, including BHK21IC13, might possibly undergo
transformation to some extent, mouse embryo primary culture cells were chosen as an
example of normal cells. The growth curve and SI levels are shown in Fig. 5. Mouse
embryo cells have a long lag phase of around 35 h. The SI of cells from confluent
monolayers is 0-25; it then increases during the lag phase and reaches the maximum
value of 4-0 at about 24 h. Shortly before the beginning of the exponential phase the
index begins to fall gradually. This changing pattern and the actual value of the SI are
Cytotoxic effect of oxidized spermidine
79
very similar to those of BHK21JC13. In this respect therefore the BHK21JC13 cells
retain the normal character. As expected, mouse L cells, which were established as a
permanent line a long time ago, showed a quite different SI pattern. L cells start
growing exponentially right after seeding with virtually no lag phase, although their
generation time is almost identical with mouse embryo cells in the exponential phase
(Fig. 6). At confluence they possess the characteristic high SI of transformed cells
and the maximum value attained is slightly lower than that of the mouse embryo cells.
Two new cell lines have been established from mouse embryo cells in this laboratory
by Dr J. F. Williams. Both lines came from the same batch of mouse embryo primary
culture. During passage the C 3 line was always kept at low cell density while the Cio
4-X10 6 r-
10
2x10 6
10
« 2x10'
106
10*
Z 5x10=
o
Z 5x10*
10 20 30 40 50 60 70
Incubation time of initial culture, h
Fig. 5
I
I
I
I
10 20 30 40 50 60 70
Incubation time of initial culture, h
Fig. 6
Fig. 5. Change in SI of mouse embryo primary culture cells in various growth
phases. • , number of cells per dish; O, SI.
Fig. 6. Change in SI of mouse L cells in various growth phases. • , number of cells
per dish; O, SI.
line was allowed to grow to high cell density. As a result of this selection procedure
the C 3 line stops growing at low cell density, while the Cio line grows to form a thick
monolayer (J. F. Williams, unpublished data). The SI values are shown in Table 4.
The maximum SI of both lines are similar to mouse embryo primary culture cells or
L cells. The C3 line shows a high SI at confluence comparable to that of L cells, while
the Cio line is more similar to embryo primary culture cells. Hence by the criterion of
SI the C3 line, which stops growing at very low cell density, behaves like transformed
cell lines, while the Cio line, which forms a thick monolayer, behaves like normal
cell lines.
In Table 4 data for various cell lines are shown. Primary embryo cultures as well as
human WI-38 have low SI values. On the other hand those lines which have been
maintained in laboratories for a long time show very high SI both at confluence and
at 15 h.
80
H. Otsuka
DISCUSSION
The data presented in this paper indicate that normal cells grown to confluence are
very sensitive to the cytotoxic effect of oxidized spermidine while polyoma virustransformed cells, adenovirus type 12 transformed cells or spontaneously transformed
cells are considerably more resistant.
Table 4. SI of various cell lines
Cell line
Mouse embryo primary culture
Rat embryo primary culture
Chick embryo primary culture
Human WI-38
L-cell
C3 mouse cell line
Cio mouse cell line
Polyoma-tran8formed rat cell line (RPTi)
Adenovirus type 12 transformed
rat cell line (RTi)
Human liver cell line
HeLa
Hep-2
BSC-i
Type
Fibroblastic
Fibroblastic
Fibroblastic
Fibroblastic
Epithelial
Epithelial
Fibroblastic
Epithelial
Epithelial
Epithelial
Epithelial
Epithelial
Epithelial
SI
at confluence
0-25
SI
at 15 h
3-0, 4-0
20
0-25
1-5, 2-0
2-5. 3-o
2'5> 3'0
O'S, i'O
3-5
15
7-0
4-5, 4'5
4'O, 4'5
40, 4'O
i-5
2-5,
3-o,
4'S,
4'O,
S-o
3-o
3-S
S-o
5-°
15
8-o
125, 150
12-5, 12-5
60, 70
Both BHK21IC1J cells and embryo primary culture cells obtained from confluent
cultures have a very long lag time before they enter exponential growth (Figs. 3, 5).
This long lag time could be a consequence of their low SI at high cell density. The
relationship between the growth curve and the SI curve might suggest that the growth
of these cells is initiated only after the SI has attained a certain level. By contrast
transformed cells or other permanent cell lines, which possess a higher SI to start with,
grow virtually without any lag.
Since the mechanism of action of the cytotoxic agent produced by the oxidation of
spermidine is not fully understood, it is difficult to know what kind of properties of
transformed cells are associated with higher spermidine index. Changes in the cell
surface have been postulated as the basis for the transformed phenotype (Abercrombie
& Ambrose, 1962; Hakomori & Murakami, 1968). It has been reported that virustransformed cell lines react with an agglutinin, while under identical conditions their
untransformed parent cell lines do not (Aub, Tieslau & Lankester, 1963; Burger &
Goldberg, 1967). Since a short treatment with a low concentration of protease exposed
agglutinin receptor sites in the parent cell line indistinguishable from the sites in
transformed cells, it was proposed that tumour virus converts the membrane of the
parent cells in such a fashion that the agglutinin receptor sites become exposed and
available to the agglutinin (Burger, 1969). However, this explanation cannot account
for the high SI because the levels reported were all obtained using trypsinized cells.
Moreover, spermidine tests carried out on BHK21JC13 cells that were not trypsinized
gave SI levels twice as high (author's unpublished data).
Cytotoxic effect of oxidized spermidine
81
A high SI of cells at confluence is associated with ability to grow in soft agar suspension. Polyoma transformed cells, BHK.2i\C i^rif^, L, HeLa and Hep-2 cells have
been shown to grow in soft agar while primary embryo cells and BHK2i/Cij cells
do not grow under these precise conditions. However, BHK21JC13 cells can grow
and form colonies in soft agar suspension, if the conditions are controlled so that a high
SI is maintained. This can be achieved for example by inoculating large cell numbers
(around 5 x ioB cells per ml of soft agar medium), or by obtaining the cells from monolayers in lag or early lag phase, when the SI is highest: numerous colonies of BHK21J
C/j cells are then observed in the agar after incubation for 7 days. Montagnier (1968)
reported that a 'conditioned medium' obtained from confluent BHK21JC/j cultures
helped the same cells to grow in agar suspension. My preliminary data suggests that
a factor which neutralizes the cytotoxic effect of oxidized spermidine is present in the
'conditioned medium'. Study of this factor is now in progress.
I am grateful to Professor J. H. Subak-Sharpe for his discussions and encouragement. I also
thank Dr J. F. Williams for his suggestion to use mouse C j and C10 lines. The excellent
technical assistance of Mrs S. Robertson is thankfully acknowledged.
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{Received 12 November 1970)
Figs. 7-10. BHK21IC13 cells treated with o (control), 0-5, 1 'O, and 1 -5 /tg of spermidine, respectively. The photographs were taken after staining with Giemsa. In
routine assays the cytotoxic effect was examined without staining, x 132.
Cytotoxic effect of oxidized spermidine
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