J. Cell Sci.

ia, 655-664 (1973) 655

Printed in Great Britain

SOME EFFECTS OF ACTINOMYCIN D,

CYCLOHEXIMIDE AND PUROMYCIN ON
CELL ADHESION

L. WEISS AND M. K. CHANG

Department of Experimental Pathology, Roswell Park Memorial Institute,
Buffalo, N.Y. 14203, U.S.A.

SUMMARY
The metabolic inhibitors cycloheximide, puromycin and actinomycin D, increased the rate
of adhesion of Ehrlich ascites tumour cells to coverslips and plastic surfaces, over periods of
up to 2 h in vitro. Among a number of possible explanations of this effect is a decreased pro-
duction of hypothetical anti-adhesion factor(s). It is suggested that the difference between the
increased rate of adhesion observed by us, and the decrease or lack of effect of the drugs
observed by others, is related to prior trypsin-treatment of cells by the other observers.

INTRODUCTION
In spite of the many studies made over the last decade, the basic mechanisms
involved in cell adhesion are still largely unknown. Some indication that active cell
metabolism is essential for cell adhesion to occur comes from the work of Moscona &
Moscona (1963) and others, which indicates that actinomycin D, puromycin and
cycloheximide inhibit the reaggregation of dissociated embryonic cells. However, in
the case of Ehrlich ascites tumour cells adhering to coverslips, it has been shown
(Weiss, 1972) that metabolic inhibitors such as the cardiac glycoside ouabain, enhance
rather than retard, the rate of cell adhesion.
In this communication, we describe the effects of actinomycin D, cycloheximide
and puromycin on the rate of adhesion of Ehrlich ascites tumour cells to glass and
plastic surfaces in vitro, and attempt to account for the apparent contradictions
between our results and those of others.

MATERIALS AND METHODS

Cell culture
Ehrlich-Lettre hyperdiploid ascites carcinoma (EAT) cells were grown at 37 °C in suspension
culture at densities of 2-6 x io'/ml, in RPMI medium 1630 (Moore, Sandberg & Ulrich, 1966)
supplemented with calf serum. Cell viability was assessed during the various experiments by
either standard trypan blue exclusion tests or by measuring reproductive integrity in vitro.
656 L. Weiss and M. K. Chang

Reagents
The effects of metabolic inhibitors on cell adhesion were determined in the presence of final
concentrations of o-oi to 10/Jg/ml cycloheximide, io~* to io~7 M puromycin, and 1/ig/ml
actinomycin D. Cells were routinely incubated with the reagents for 30 min at 37 °C before
'starting' the experiments.
In some experiments, cells were incubated in 0-2 % trypsin (Difco 1:250 in Ca, Mg-free
phosphate-buffered saline at pH 7-6—7-8; io7 cells/5 ml trypsin solution) for 25 min at 37 °C.
They were then washed 3 times and then suspended in serum-containing medium, and cyclo-
heximide in final concentrations of o-i or i-o /Jg/ml was added. The cell suspensions were then
treated in a similar manner to non-trypsinized cells.

Cell adhesion
The rate of cell adhesion to (a) glass coverslips and/or (b) plastic surfaces was determined as
follows:
(a) 2-5-cm diameter coverslips were placed in polyethylene dishes of 28 cm diameter, and
covered with 2 ml of suspension containing 5 x io4 viable cells per ml. The individual dishes
were sealed and incubated at 37 °C, and at given times the non-adherent cells were washed off
the coverslips, by gently dipping them twice, in each of 2 beakers of medium maintained at
37 CC. The coverslips were inverted on to microscope slides, and the cells adherent to 8 randomly
selected areas of 0-39 mm ! around their centres were counted, using an eyepiece graticle, in a
phase-contrast microscope.
(b) Single drops of the cell suspensions, containing approximately 340 cells in a volume of
0-0068 ml, were delivered into each well of 'Microtest' plates (Falcon Plastics, Los Angeles,
Calif.), through 25-gauge needles attached to i-ml syringes. The polystyrene plates contained
60 numbered wells, with flat circular bottoms of 1-5 mm diameter. Lids were put on individual
plates which were incubated at 37 °C in an atmosphere of 5 % COS in air. At stated times, the
plates were inverted, and the residual adherent cells counted using phase-contrast microscopy.
In both techniques, for purposes of comparisons between experiments, the cell numbers on
the different surfaces were expressed as percentages of the mean of the control in each particular
series. After 60 min, in the case of the coverslips, 100% is equivalent to approximately 60—75
cells, and in the 'Microtest' plates, 100% = 150—200 cells.
14
C incorporation
The various reagents were added to centrifuge tubes, containing 20-ml aliquots of suspensions
of 5 x io 5 cells per ml in culture medium, and incubated for 30 min at 37 °C. To each tube was
added 10 /*Ci of L-uC-amino acid in a volume of 0-2 ml (New England Nuclear, Boston, Mass.:
NEC-445 mixtures containing 15 individual 14C-amino acids in 01 N HC1). After 5, 60 and
120 min incubation at 37 °C, the samples were washed twice by centrifugation, in medium
containing o-i % casein hydrolysate, then mixed with 2 ml of 10% trichloroacetic acid and
kept at 4 °C for 15 min. The suspensions were run through 13-mm Millipore filters (8 /im pore
size), which were then placed in liquid scintillation counting vials, to which were added 1 ml of
10 N ammonium hydroxide, followed by 10 ml of Bray's solution. Radioactivity was measured
over 3-min periods in a liquid scintillation counter.

Electrophoretic mobilities
These were measured at 37 °C with cells suspended in culture medium, in a cylindrical tube
apparatus with sintered, grey platinum electrodes. Measurements were made on unwashed
cells in the presence and absence of cycloheximide (1 /tg/ml), and on cells incubated in the
presence of cycloheximide and then washed twice and resuspended in medium.
 Metabolic inhibitors and cell adhesion 657

Table 1. Effect of cycloheximide on the adhesion of EA T cells to coverslips after 1 and

2 h culture, expressed as percentages of the means of controls

Final cyclo-
heximide con- % Adherent cells ± S.E. (no. observations) after
centration,
jig/ml 1h 2h

0 (control) 100 ± 1-36 (105) 100 ± 1-54(93)

i-o I22-I ±2-49 (H3) 118-812-34(90)
o-i 1147 ±4-86 (20) 127-3 ±4'33 (22)
o-oi 98-6 ±3 59 (20) m-4±2-6s (21)

Table 2. Effect of washing cells preincubated with cycloheximide, on their

adhesion to coverslips after 1 and 2 h incubation

Preincubation with %Washed adherent cells ± S.E.

cycloheximide in (no. observations) after
final concentration,
/ig/ml 1h 2h
0 (control) ioo-o±4 •63 (10) 100013-91 (10)
i-o 97-2 ±6 •72 (10) 106-815-62 (10)
o-i 96-7 ± 5•88 (10) 1036 ±3-53 (10)
o-oi 979 + 2•85 (10) 105-8 ±4-42 (10)

RESULTS
Rate of adhesion to glass
Cycloheximide treatment. The results shown in Table 1 indicate that the rate of
adhesion of EAT cells to coverslips during 60 min incubation is significantly in-
creased (P < o-oi) by cycloheximide in concentrations of 1 and o-i /tg/ml. After 2 h
incubation, the rate of adhesion is increased in the presence of i - o, o-i and o-oi //•g/ml
cycloheximide.
A variant of this group of experiments was one in which cells were pre-incubated
for 30 min with different concentrations of cycloheximide, washed (x 3) and re-
suspended in medium, then incubated over coverslips for periods of 1 or 2 h. The
results of one such experiment summarized in Table 2, indicate that washing cyclo-
heximide-treated cells restores their rate of adhesion to that of the controls.
The results summarized in Table 3 show that after incubation with trypsin,
cycloheximide in final concentrations of o-1 and 1 -o fig/m\ had no significant effect
on the rate of cell adhesion over 1 h, compared with trypsin-treated cells incubated
in the absence of cycloheximide.
Puromycin treatment. The results shown in Table 4, indicate that at final concentra-
tions of io~B and io~6 M, puromycin significantly increases the rate of cell adhesion,
as assessed after 60 min. After 2 h, an increased rate is observed with concentrations
up to io~~4 M. Concentrations lower than io~6 M do not produce an obvious effect on
the rate of cell adhesion, during 2 h.
658 L. Weiss and M. K. Chang

Table 3. Effect of cycloheximide on the adhesion of trypsin-treated EAT

cells to Microtest plates, after 1 h incubation

Final concentration % Adherent cells ± S.E.

cycloheximide, /tg/ml (no. observations)
0 (control) 1000 ±1-85 (66)
01 9 8 0 ± 1 4 9 (63)
i-o 105-3 ±2-56 (48)

Table 4. Effect of puromycin on the adhesion of EAT cells to coverslips

% Adherent cells ± s.E. (no. observations) after

Final puromycin , *- v
concentration, M 1h 2h
o (control) 100 ±2-74 (64) 100 ±1-81(64)
io-» 98-21411(24) 95-412-94(24)
o-8 > P > 0-7 0 4 > P > 0-3
io~* 10912-40(64) 108-712-54(64)
0-02 > P > o-oi o-oi > P > o-ooi
IO~B 1 1 4 6 ± 2-o6 (80) I I 2 - O ± I-gg (80)
P < o-ooi P < 0001
4
10- 97'3±3'98(44) 106-512-85(44)
o-6 > P > 0-5 0-05 > P > 0-02

Table 5. The adhesion of EAT cells to Microtest plates after 1 h

incubation in the presence of cycloheximide

Final concentration % Adherent cells ± S.E.

cycloheximide, /tg/ml (no. observations)

0 (control) IOO-O± 2-27 (48)

o-oi II9-3 ±5'54 (22)
o-i 110-613-96(35)
o-5 118-113-78(24)
i-o "7-5 ±4-52 (24)
2-O 108-014-59 (24)
SO II3-3 ±435 (24)
IO-O 88-012-57(47)

Actinomycin D treatment. In final concentrations of 1 /tg/ml, actinomycin D was

without demonstrable effect on the rate of cell adhesion, up to 60 min. Expressed as
percentages of the mean of the controls, 100 ±2-03 (56) of the controls adhered to
the coverslips, compared with 99-5 + 1-75 (66) in cultures treated with actinomycin D.
After 2 h incubation 111-3 ± 2-3 (53) adhered, compared with the controls of 100 ±
2-1 (52). This increase is highly significant (P < o-oi).
 Metabolic inhibitors and cell adhesion 659

Rate of adhesion to Microtest plates

The results shown in Table 5 show that the rate of cell adhesion, assessed at
60 min, is increased by cycloheximide in final concentrations in the range of o-oi-
5/tg/ml; at concentrations of 10/ig/ml, there is a significant reduction (P < o-oi).

0 10

Fig. 1. Effects of cycloheximide (concentrations given in fig/m\) on 14C-amino acid

incorporation by EAT cells after 10, 60 and 120 min incubation. O, controls, con-
taining no cycloheximide.
Fig. 2. Effects of puromycin (concentrations given in M) of 14C-amino acid incorpora-
tion by EAT cells after 10, 60 and 120 min incubation. O, controls, containing no
puromycin.

14
C incorporation studies
As shown in Fig. 1, there are consistent proportionate falls in the amounts of
14
C-amino acid incorporation, over the cycloheximide concentration range of o-oi-
10/fg/ml.
Puromycin treatment. As shown in Fig. 2, puromycin produces definite decreases in
amino acid incorporation in final concentrations of io~4 and io~6 M; IO~6 and io~7 M
puromycin result in slight reductions which are indistinguishable from each other.

Viability tests
In one series of experiments, cells from one spinner culture were equally divided
between 2 spinner flasks. Cycloheximide in a final concentration of 1 /*g/ml was added
to one flask. After 2 h incubation, the contents of both flasks were washed 3 times,
resuspended in medium, and returned to the spinner flasks. Samples were taken
for cell counts and trypan blue exclusion tests for times up to 2 days after washing.
66o L. Weiss and M. K. Chang

600 -

400 -

o
X
01
U
200 i

Fig. 3. Cells from a common pool were incubated in the presence (O) of 1 /Jg/ml
cycloheximide, or in its absence (•) for 2 h, washed and returned to spinner flasks.
Viable cell counts were made (ordinate; cells/ml) at the indicated times after washing.
Allowing for the slight differences in initial inocula, no differences are detectable after
50 h.

No significant difference in the percentage viability (90-97%) was observed, and the
growth patterns were very similar (Fig. 3). Variants of this experiment also failed to
reveal loss of viability following up to 2 h exposure to cycloheximide until concentra-
tions as high as io/^g/ml were used. Following exposure to this dosage for 2 h, cells
were washed and returned to spinner cultures. After 45 h, without replenishment of
medium, the viability had fallen to 52% compared with 87% in the controls.

Electrophoretic mobilities
Compared with the control value of — I-IO ± 0-03 (61 observations) /tra s~x V"1 cm,
cells measured in the presence of 1 /£g/ml cycloheximide had values of —1-04 ±0-02
(72), and cells exposed to the drug, and then washed, had mean mobilities of —1-09 +
0-02 (86). These values were not significantly different from the controls.

DISCUSSION
In the present work we have attempted to isolate part of the problem of adhesive
mechanisms by studying one parameter, the rate of cell adhesion to 2 specified, non-
living surfaces in vitro. In these experiments, we are not really studying cell adhesion
to glass or plastic surfaces, but rather adhesion to the proteinaceous and other
materials originating from the cells and their culture media, which adsorb to the sur-
faces. Therefore, from a mechanistic viewpoint, studies of the present type may well
provide useful insight into cell-to-cell adhesion.
The physicochemical nature of its periphery partially determines the contact inter-
 Metabolic inhibitors and cell adhesion 661
actions of a cell with any surface. Therefore, as this region of the cell is in a dynamic
state, it is expected that metabolic inhibitors will affect both the nature of the cell
periphery, and various cell contact phenomena. Thus, using cultures of reaggregating
neural retina cells from trypsin-dispersed chick embryos, Moscona & Moscona (1963)
demonstrated that both puromycin and actinomycin D inhibited cell adhesion. These
observations on cells from embryos have been repeated and extended by other workers
(Richmond, Glaeser & Todd, 1968; Dunn, Owen & Kemp, 1970). In addition, the
involvement of endogenous ATP in cell adhesion is suggested by Michaelis &
Delgarno (1971).
The present results differ from those expected from the work on embryonic cells,
in that the rate of adhesion of Ehrlich ascites tumour cells to the surfaces used here,
is enhanced by both puromycin and to a lesser extent, actinomycin D. The adhesion-
promoting effect of puromycin is associated with reduced incorporation of amino
acids. However, these effects of puromycin do not in themselves establish a causal
relationship between cell adhesion and protein synthesis, because of the additional
effects of the drugs on other metabolic products, including carbohydrates (Dunn et al.
1970).
In the presence of final concentrations of cycloheximide up to 1 /tg/ml, which
effectively inhibits protein and RNA synthesis (Tata & Widnell, 1966), the rate of
cell adhesion was also enhanced, in the absence of permanent cell damage. At con-
centrations of 10/ig/ml, cycloheximide reduced the rate of cell adhesion; however,
these results are associated with overt cytotoxicity and cannot be usefully discussed
in the present context.
The effects of cycloheximide in increasing the rate of cell adhesion were abolished
by washing cycloheximide-treated cells before starting the adhesion experiments.
This well known reversal of the effects of cycloheximide by washing parallels many
observations on protein synthesis. The fact that the adhesion-potentiating activity of
actinomycin D is much less marked than that of cycloheximide also possibly indicates
a correlation between inhibition of protein synthesis and increased adhesion, since it
is recognized that small quantities of RNA may be synthesized in mammalian cells in
the presence of actinomycin D (Stern & Friedman, 1970).
First, we will comment on the unexpected enhancement of adhesion by cyclo-
heximide, puromycin and actinomycin D, and secondly we will discuss why our
results should differ from the effects observed with embryonic cells.
The adhesion of cells to coverslips and metal surfaces has been associated with the
exudation of proteinaceous material from cells (P.Weiss, 1945; Rosenberg, i960;
Poste, 1970), and the release of proteinaceous materials from EAT cells while they
were in the process of adhering to coverslips and cellular monolayers has been demon-
strated by Maslow & Weiss (1972). It has also been shown that proteins originating
from cultured cells enhance cell adhesion (Daday & Creaser, 1970), and Poste &
Greenham (1971) have shown that synthesis of cell 'coat' materials at the cell/sub-
stratum interface is inhibited by puromycin. It would therefore be expected that
inhibitors of protein synthesis will retard cell adhesion rather than accelerate it, as
presently observed.
662 L. Weiss and M. K. Chang
It is well established that in common with other organelles, the peripheries of cells are
in a dynamic state with considerable turnover (Warren & Glick, 1968; Garner, Glick
& Warren, 1970). It is possible, therefore, that inhibition of synthetic processes leads
to an abnormal cell periphery, which has physicochemical characteristics favouring an
increased rate of adhesion. However, the electrophoretic mobility measurements do
not support this suggestion, although abnormalities could occur without changes in
mobility; and studies on adhesion rates do not in themselves indicate whether changes
in rate are accomplished by ' normal' or ' abnormal' mechanisms.
Another possibility is that cells normally produce anti-adhesion factors, and that
by interfering with the synthesis of these factors, inhibitors of protein synthesis
increase the rate of cell adhesion. In addition to preventing the initiation of adhesions,
such factors could possibly be involved in non-lethal autolysis of the cell periphery
(Weiss, 1967) and tend to destroy cell/substratum contacts as they are formed. The
hypothetical anti-adhesion factors are not, on present evidence, considered to play an
all-or-none role in preventing cell adhesion, but rather to exert a rate-regulatory
effect which is counterbalanced by adhesion-promoting factors. The anti-adhesion
factors discussed here, are distinct from the serum factors proposed by Curtis &
Greaves (1965), which are apparently destroyed by cell metabolic products, since, by
inhibiting this destruction, the metabolic inhibitors used in the present work would
be expected to inhibit rather than enhance adhesion.
There are a number of possible reasons why the present results appear to be at
variance with many of the results obtained by others on reaggregating embryonic
cells, quite apart from the obvious inherent differences between for example, neural
retina cells from chicken embryos, and cells derived from, and capable of initiating
adenocarcinomata in mice.
The reaggregation of embryonic cells in vitro to form histotypic structures involves
a complex series of events occurring after initial contact has been made between cells,
and there is no doubt that inhibitors of protein synthesis interfere with these recon-
structive processes. However, in short-term experiments with embryonic material,
such as those reported by Lilien (1968), cycloheximide (2/ig/ml) had no detectable
effect on the rate of cell adhesion for periods of up to 2 h. In the present experiments
cycloheximide produced an approximate 20 % increase in cell adhesion after 2 h
culture, and the problem is to account for differences within this time-scale.
Cells are routinely isolated from embryonic chick organs by incubation with trypsin,
which is known to affect their surfaces (Barnard, Weiss & Ratcliffe, 1969) and sub-
sequent adhesive behaviour (Weiss & Kapes, 1966). The cells are then placed in an
environment which is not only chemically different from that in ovo, but is one in
which their normal spatial interrelationships with each other are at least temporarily
disturbed. In contrast, the tumour cells used by us are simply taken from a spinner
culture, where they are adapted to the in vitro conditions, and their rates of adhesion
are assessed under similar environmental conditions. We suggest that these differences
in pretreatment are partly responsible for the different responses of the cells to
metabolic inhibitors. This suggestion is supported by the observation that following
trypsin treatment, the rate of adhesion of EAT cells to Microtest plates is no longer
 Metabolic inhibitors and cell adhesion 663
increased by cycloheximide. Additional support is provided by experiments showing
that inhibitors of protein synthesis were without apparent effect on the readhesion of
trypsinized 3T3 cells to plastic surfaces of the type used here (Kolodny, 1972), and
by the observation of Weiss & Maslow (1972), that if trypsin-dispersed chick neural
retinal cells are given a 24-h 'recovery' period before reaggregation is allowed to
occur, then their adhesion is far less sensitive to cycloheximide-inhibition over 24 and
48 h than in cells given no recovery period. In our opinion, this common previous
history of trypsin-dispersion is an often overlooked factor in experiments designed to
determine the effects of metabolic inhibitors.
This work was partially supported by Grant BC-87E from the American Cancer Society Inc.

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{Received 12 June 1972)