CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
ACTINONYCIN D STIHULATION OF
I\
TERA'l'Oi.ffi CELL ADHESION
A thesis submitted in partial satisfaction of the
requirements for the degree of Master cf Science in
Biology
by
Robert Ray Nystrom
June,
197,8
- - - ------------------------
------
·rhe Thesis of Robert Ray Nystrom is approved:
Dai~ A.0Kuhn, Ph.D.,.
'Richa_rg L. Potter, Ph.D.
Ste~r~~- B.. Opp~hheimer, Ph.D.
California State University, Northridge
ii
ACKNOWLEDGMENTS
I r.vould like to extend my thanks and
appreciatio.n to Dr. Steven B. Oppenheimer for
his advice and encoui'agement during the research
and preparation of this thesis.
I would also
like to thank Dr. Daisy A. Kuhn and Dr. Richard
L. Potter for their assistance and support.
iii
-
--..,....--~
-~-----
-~~---
-~-~--
-~
-~~-
--
-~-
TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS .
. .. .
iii
. . . . .. .
LIST OF TABLES
LIST OF FIGURES .
iv
vi
ABSTRACT
vii
INTRODUC'riON
1
MATERIALS AND METHODS .
8
Teratoma tissue cultures
8
Intercellular adhesion assay
8
Glutamine synthetase extraction .
11
Glu·tamine synt.hetase assay
12
RESULTS
~
15
•
Intercellular adhesiveness of
cultured teratoma cells . .
15
Specific activity of glutamine synthetase
from cultured teratoma cells
. . .
19
DISCUSSION
21
REFERENCES
26
iv
_______ .:._ - -
----------
..:__
_________ ____ _
..;__
LIST OF TABLES
Page
Table
1.
2.
Effect of actinomycin D on intercellular
adhesion of cultured teratoma cells
Effect of actinomycin D on GS specific
activity of cultured t.eratoma cells . .
v
16
.
. .
20
LIST OF FIGURES
Figure
1.
Page
Model for mechanism of hydrocortisone
and actinomycin D stimulation of GS
activity
. . . . • • . . . .
. . .
5
. 10
2.
Adhesion assay
3.
Glutamine synthetase extraction and
assay . . .. . . . . . . . . •
. .
.
.
.
.
"' 14
Typical reaggregation pattern of
cultured teratoma cells during an
adhesion assay
. . . . . . . . . • .
.
.
.
.
. 18
4.
vi
ABSTRACT
ACTINOMYCIN D STIMULATION OF
TERATOMA CELL ADHESION
by
Robert Ray Nystrom
Master of Science in Biology
In cultured mouse teratoma cells, actinomycin D has
been shown to stimulate glutamine synthetase (GS) specific
activity with a concomitant increase in intercellular
adhesion.
Cultured mouse teratoma cells were preincubated
for 24 hours with 5
~g/ml
actinomycin D.
They were then
dissociated.and incubated for 1 hour on a gyratory shaker.
The decrease in single cell number, as determined with an
electronic particle counter, was used to calculate the percent adhesion.
The GS specific activity of these cells was
then determined by an assay involving the glutamyltransferase reaction.
It was found that the actinomycin D
treated cells show.ed a 31.4 + 9. 9% [mean + standard error
(S.E.)] increased intercellular adhesion and a 53+ 17%
increase in GS specific activity.·
vii
The increased adhesiveness of these cells may be
due to the stimulated GS specific activity and subsequent
synthesis of L-glutamine, which has been shown by Oppenheimer et al.
(1969) and Oppenheimer (1973) to be required
for teratoma intercellular adhesion.
They suggest that
the L-glutamine is required for the synthesis of complex
cell surface carbohydrates involved in intercellular adhesion of teratoma cells.
viii
INTRODUCTION
There is strong evidence that L-glutamine plays a
key role in intercellular adhesion.
Oppenheimer et al.
(1969; 1973) have shown that L-glutaminc, which is involved
in the synthesis of complex carbohydrates, is required for
the intercellular adhesion of mouse ascites teratoma cells.
Also, they have suggested that L-glutamine promotes adhesion in these cells by transaminating fructose-6-phosphate
to glucosamine-6-phosphate, a metabolic intermediate in the
synthesis of these complex carbohydrates.
Since L-glutamine seems to play an integral role in
teratoma intercellular adhesion, its synthesis and regulation should be considered further.
There seems to be only
one pathway for the synthesis of L-glutamine, although
others have been investigated and cannot be conclusively
eliminated (Meister, 1962).
This pathway involves the
formation of L-glutamine from glutamate and ammonia through
the action of the enzyme glutamine synthetase [L-glutamate
ammonia ligase (EC 6.3.1.2)].
Glutamate + NH3 + ATP
The same enzyme has
al~o
----+
Glutamine + ADP + Pi
been shown to catalyze the glu-
tamyltransferase reaction (Levintow et al., 1955).
Glutamine + Hydroxylamine
~ y -
1
Glutamylhydroxamate + NH3
2
Y - Glutamylhydroxamate gives a characteristic brown color
with ferric chloride, thus making it a convenient tool for
the detection of glutamine synthetase (GS) activity (Meister, 1962).
GS participates in many important biosynthetic
pathways, e.g., those leading to the synthesis of purines,
pyrimidines, amino acids, and diphosphopyridine nucleotides.
It also serves a significant function in the storage of
aw~onia
in the form of L-glutamine (Meister, 1962).
A variety of sources have been used for the isola-
tion of GS including the bacteria Escherichia coli (Shapiro
and Ginsburg, 1968) and Bacillus subtilis (Duel and Ginsburg, 1970), sheep brain (Meister, 1962), pea seeds and
rat liver (Tate and Meister, 1971), and chick neural retina
(Sarkar et al., 1972).
Although there is a considerable
similarity in amino acid sequence and molecular weight,
these enzymes are not physically or chemically identical.
Subunit molecular weights vary from 42,000 in neural retina
GS (Sarkar et al., 1972) to 50,000 in bacterial GS (Shapiro
and Ginsburg, 1968; Duel and Ginsburg, 1970).
The intracellular regulation of GS has been studied
in various eucaryotic systems.
If HeLa cells are grown on
a medium high in glutamic acid (20 mM), the GS specific
activity will be greatly increased, while the addition of
2 mM L-glutamine to the growth medium will cause a marked
reduction in that activity (Demars, 1958).
Depression of
3
GS activity by L-glutamine was also observed in cultured
hepatoma cells (Kulka and Cohen, 1973}.
In addition, when
these cells were transferred from a medium supplemented
with L-glutamine to a medium lacking L-glutarnine, there was
a subsequent increase in GS specific activity.
Further-
more, this increase was inhibited by cyclohexamide, indicating that protein synthesis was necessary for the increased
activity.
Similar results have been obtained with cul-
tured hamster cells (Tiemeier and Milman, 1972).
It has
also been shown, that various steroid hormones (Moscona and
Hubby, 1963; Barnes et al., 1971; Kulka and Cohen, 1973)
and actinomycin D (Moscona et al., 1972; Connolly and
Oppenheimer, 1975) can stimulate GS activity.
Moscona et al.
(1972) have proposed a model for the
mechanism of hydrocortisone and actinomycin D stimulation
of GS activity in chick neural retina (Figure 1) .
The
model involves the interaction of at least five gene products:
1) a structural gene(s) which codes for mRNA for the
GS enzyme; 2) a repressor gene(s) which codes for a product
that prevents the transcription of the GS mRNA and the
desuppressor mRNA; 3) a suppressor gene(s) which codes for
.
.
a relatively labile suppressor product that prevents the
translation of GS mRNA into enzyme; 4) a desupressor gene(s)
which codes for a product that inactivates the suppressor
gene, thus allowing the GS mRNA to be translated; 5) a
qegredation gene(s) which codes for a product that
4
Figure 1.
Model for the mechanism of hydrocortisone and
actinomycin D stimulation of GS activity in chick neural
retina as proposed and diagrammed by Hoscona et al.
(la) normal state,
(lb) hydrocortisone stimulated state,
(lc) actinomycin D stimulated state.
r
= repressor
gene product; GS
-· suppressor gene; S
=
R
=
repressor gene;
= structural
gene; DE SUPP
suppressor gene product; DEGR =
degredation gene; DG = degradation gene product; GS
tamine synthetase; HC = hydrocortisone; Act D
D.
(1972).
=
=
glu-
actinomycin
5
DNA
la.
.•...__+1----- s
t
(GS)
lb.
DG
T
GJ
mRNA
DS ...,_ S
DG
+
GS
lc.
Gs
I
silliP
I I
SUPP
,..__ _ _ _ _ _ Act D ----------~
mRNA
l
GS
6
inactivates GS.
Under uninduced conditions (Figure la),
only the repressor, suppressor and degradation genes are
active, therefore, GS mRNA transcription and GS translation
are inhibited, but some GS templates leak through and
account for the basal level of GS activity observed in uninduced cells.
When hydrocortisone is present, the repressor
gene is bound and its transcription prevented, thus freeing
the GS structural gene and desuppressor for transcription.
Also, the desuppressor gene product binds to and inactivates the suppressor gene product, and the net result is an
increase in GS activity (Figure lb).
Actinomycin D, a
transcriptional protein synthesis inhibitor, on the other
hand, binds to and prevents the transcription of all .five
gene products, but GS continues to be translated from the
relatively stable, preformed, GS mRNA templates (Figure lc).
This is possible since the suppressor gene product is relatively labile, and must be continuously transcribed to be
effective.
When the suppressor loses its effectiveness,
and no more suppressor is being produced because of the
inhibition by actinomycin D, then there is an increase in
the amount of GS that is translated from the stable preformed GS mRNA templates, and thus a net increase in GS
activity occurs.
Connolly and Oppenheimer (1975) have
shown that an increase of GS activity does indeed occur in
cultured mouse teratoma cells, when they are incubated with
actinomycin D.
Similar results have been obtained by
(
7
Weisman and Ben-Or (1970) and Rief-Lehrer (1971) with chick
neural retina cells, and by Kulka and Cohen (1973) with
cultured hepatoma cells.
Hale (1977) has shown that the hydrocortisone stimulated increase in GS activity of ascites and cultured teratoma cells is accompanied by an increase in intercellular
adhesion.
This implies some relationship between GS speci-
fic activity and intercellular adhesion of teratoma cells.
The purpose of the present study is to explore the effect
of actinomycin D stimulated GS activity on intercellular
adhesiveness in cultured teratoma cells.
t~at
It will be shown
in cultured teratoma cells, actinomycin D stimulated
GS activity, like hydrocortisone stimulated GS activity, is
accompanied by an increase in intercellular adhesiveness.
MATERIALS AND METHODS
Teratoma tissue cultures.
Cultures of mouse teratoma cells
were obtained from Dr. John Lehman, Department of Pathology,
University of Colorado Medical Center.
Stock cultures were
maintained in so-called complete minimum essential medium
(CMEM) , placed in 75 cm2 plastic tissue culture flasks
(Corning Glass Works, Corning, NY), and incubated in a
humidified 5% C02 atmosphere at 37° C.
This medium con-
sisted of Eagle's minimum essential medium with Earle's
salts (MEM)
(Grand Island Biological Co., Grand Island,
NY) , plus 10% heat inactivated fetal bovine serum (Grand
Island Biological Co., Grand Island, NY), 50 ].lg/ml each of
gentamicin (Schering Corp., Kenilworth, NJ) and fungizone
(E.M. Squibb & Sons, Inc., New York, NY), and 2 mM Lglutamine (Microbiological Associates, Bethesda, MD).
medium was changed every 2-3 days.
The
Cells were transferred
weekly in MEM supplemented with 0.2% trypsin (1:250, Difco
Laboratories, Detroit, MI) to remove cells from the flasks.
Cells were washed twice in fresh MEM, suspended in the same
.
.
medium, and reseeded into sterile flasks, each containing
10 ml CMEM.
Intercellular adhesion assay.
Twenty four hours prior to
each adhesion assay, the growth medium in 4 flasks, each
containing a monolayer of confluent cells, was replaced
8
9
with CMEM minus L-glutamine.
L-glutamine was omitted from
the medium in order to stimulate GS activity (Kulka and
Cohen, 1973).
with 5
~g/ml
Two of the four flasks were preincubated
of actinomycin D (Sigma Chemical Co., St.
Louis, MO), the others served as controls.
The control
cells were kept separate from the actinomycin D treated
cells throughout the adhesion assay, but were subjected to
the same procedure (Figure 2).
After 24 hours of prein-
cubation, the cells were trypsinized, washed in MEM, and
gently pipetted to achieve a suspension of single cells.
The actinomycin D treated cells and the control cells were
each seeded into three 100 X 15mm glass Petri dishes
(Pyrex) .
The adhesion assay was carried out in CMEM medium
without L-glutamine and fetal bovine serum.
The fetal
bovine serum was omitted from the medium because it acts as
an intercellular "glue" (unpublished).
The Petri dishes
were incubated at 37° C on a gyratory shaker at 20 rpm
(Oppenheimer and Odencrantz, 1972).
Samples of 0.2 ml were
removed every 15 minutes for 60 minutes and the number of
single cells determined with an electronic particle counter
(Model 112LT Celloscope,
P~rticl~
Data, Elmhurst, IL) by
the method of Oppenheimer and Odencrantz (1972) .
As the
cells began to aggregate, cellular adhesion was measured as
a function of the decrease in the number of single cells
(Oppenheimer and Odencrantz, 1972).
gates was determined microscopically.
The presence of aggreThe percent adhesion
10
24 hour preincubation of
cultured teratoma cells in
CMEM minus L-glutamine, with
or without 5 ~g/ml actinomycin D.
Trypsin dissociation and wash
with MEM.
0 0'0
:~±
Cells suspended in CMEM without L-glutamine and fetal bovine
serum and seeded into Petri
dishes.
Petri dishes rotated on a
gyratory shaker. Samples of
0.2 ml removed every 15 minutes
for 60 minutes and the number
of single cells determined on
an electronic particle counter.
Petri dishes scraped with a
neoprene policeman to remove
cells.
Cell suspensions collected for
subsequent enzyme extraction
and assay.
u
Figure 2.
Adhesion Assay
11
was calculated as the percent of initial cells having
aggregated after 60 minutes, and percent increased adhesion
in comparison to the controls was calculated as follows,
according to Hale (1977):
% increased
adhesion
=
% adhesion
% adhesion
treated cells
control cells
% adhesion
control cells
X 100
To insure that the disappearance of single cells
represented aggregation and not, for example, lysis, the
viability of all cells was determined before and after each
adhesion assay by the trypan blue dye exclusion method
(Roth et al., 1971).
Glutamine synthetase extraction.
After each adhesion assay
the cells were collected by scraping the 3 Petri dishes
containing the actinomycin D treated cells and the 3 Petri
dishes containing the control cells with a neoprene policeman and centrifuging the cell suspension separately for 5
minutes in 15 ml conical centrifuge tubes at 1000 rpm in
an International clinical centrifuge (rotor # 221) .
The
supernatants were poured off and the cells resuspended in
3 ml of cold phosphate buffer (0.01 M, pH 7.1) so that one
suspension contained the actinomycin D treated cells and
the other the control cells.
The cells were disrupted while
in an ice bath by three 30-second long sonications at a setting of 70 watts with an Ultratip Labsonic System equipped
12
with a Me Microtip (Lab-Line Instruments, Inc., Melrose
Park, IL}.
The sonicates were centrifuged at 12,000XG for
30 minutes at 5° C in a Sorvall RC2-B centrifuge (SS-34).
The supernatants from this centrifugation contained the
soluble GS extract.
The enzyme was stored at 4° C and
assayed within an hour of preparation.
Glutamine synthetase ·assay.
GS was assayed by the glut-
amyl transferase assay (Levintow et al., 1955; Moscona and
Hubby, 1963).
The procedure was that of Moscona and Hubby
{1963), with the following modifications.
To each reaction
tube was added 0.8 ml of a solution containing 2
NaH2P04, 2
~moles
~moles
MnCl2, 180
~moles
sodium acetate (pH 5.4).
~moles
L-glutamine, and 100
To this solution were
added 0.7 ml of enzyme extract and 0.2 ml of 0.5 mM adenosine triphosphate (Calbiochem, Los Angeles, CA) in 0.01 M
phosphate buffer (pH 7.1).
This mixture was incubated for
10 minutes at 37° C, whereupon 0.2 ml of 0.15 rnM hydroxylamine hydrochloride (Sigma Chemical Co., St. Louis, MO) was
added to each tube.
The mixture was incubated for 60 min-
utes at 37° C, at which time the reaction was stopped by
the addition of 1.5 ml of
~
solution of equal parts of 2.5
N HCl, 15% trichloracetic acid, and 5% ferric chloride in
·0.1 N HCl.
The mixture was centrifuged for 5 minutes at
1700 rpm in an International clinical centrifuge.
The opti-
cal density of the supernatant was determined at 500 nm in a
quartz spectrophometer cells (Lightpath Cells, Inc., St.
13
Louis, MO) with a visible, digital reading spectrophotometer (Beckman Model 24, Beckman Instruments, Inc., Fullerton,
CA) •
Samples were read against identical assay mixtures
with distilled H20 substituted for hydroxylamine hydrochloride.
Duplicate experimental mixtures were set up for all
assays.
Glutamylhydroxamic acid (GHA) formation was deter-
mined by comparison with a standard curve prepared with GHA
(Sigma Chemical Co., St. Louis, MO) as the standard.
Pro--
tein concentration was estimated by the method of Lowry et
al.
(1951) using bovine serum albumin (Sigma Chemical Co.,
St. Louis, MO) as the standard.
Specific activity was
defined as moles GHA formed per hour per mg of protein.
Figure 3 summarizes the procedure for the extraction of and
assay for GS.
l
14
cell suspension
from adhesion
assay
Extraction:
centrifuge (1000 rpm)
supernatant
(discard)
cells
sonicate,
centrifuge (12000XG)
·' supernatant
(enzyme extract)
Assay:
cell dleb:cis
(discard)
solution of NaH2P04, MnCl2,
Na acetate, L-glutamine
Na tartate,
CuS04•SH20,
Na2C03 in
0.1 N NaOH
ATP
incubate 10 min
at 37° C
incubate 15 min
at room temp
lI
Phenol _
reagent
incubate 60 min
at room temp
•
hydroxylamine HCl
l.ncubate 60 min
at 37° C
I
determine
optical density
at 660nm
supernant,
determine optical
density at SOOmn
Figure 3.
~
stop reaction with FeCl3,
HCl, trichloroacetic acid
centrifuge (1700 rpm)
precipitate
(discard)
Glutamine Synthetase Extraction and Assay
RESULTS
Intercellular adhesi ve·ness of cultured teratoma cells.
With the exception of one experiment, the cells preincubated
with actinomycin D consistently showed an increase in percent adhesion over the control cells.
On the basis of 10
experiments, the control cells showed a percent adhesion
of 37
~16%
[mean + standard error (S.E.)], while the actino-
mycin D treated cells showed a percent adhesion of 55 +
17% S.E.
(Table 1).
From these experiments, an increased
adhesion of 53 + 17% S.E. was calculated for the actinomycin D treated cells over the control cells (Table 1) .
The
exception was experiment 4, in which a decrease in the percent adhesion of the actinomycin D treated cells was observed.
The typical reaggregation pattern that occurred
during an adhesion assay is shown in Figure 4.
The great-
est increase in percent adhesion, for both the actinomycin
D treated and control cells, was always seen within the
first 15 minutes.
The percent of viable cells, for both
the control and actinomycin D treated cells, as determined
.
.
by the ability to exclude trypan blue dye, was between 75 85% both before and after the adhesion assay.
This indi-
cates that the calculated percent adhesion was due to aggregation and not due to cell lysis during the adhesion assay.
15
16
Table 1.
The effect of actinomycin D on the intercellular
adhesivness of cultured teratoma cells.
The cells were
preincubated for 24 hours with and without 5
mycin D.
~g/ml
actino-
Adhesion was measured after one hour incubation
on a gyratory shaker.
Percent increased adhesion was cal-
culated as described in the text.
Experiment
Number
% Adhesion
Actinomycin D
Control
Treated Cells
Cells
%
Increased
Adhesion
1
69
74
7
2
33
48
45
3
56
74
32
4
64
46
-28
5
22
35
60
6
30
34
13
7
32
76
138
8
27
52
93
9
29
41
41
10
20
45
125
37 + 16%
55 + 17%
Mean +
Standard
Error
53 +17%
17
Figura 4.
Typical reaggregation pattern of cultured tera-
toma cells during an adhesion assay (experiment 9).
The
cells were preincubated with (•) and without (o) actinomycin
D for 24 hours, dissociated by treatment with trypsin, and
incubated on a gyratory shaker for 1 hour.
The percent
adhesion was measured as described in the text.
The gluta-
mine synthetase specific activity (GS) for this experiment
was determined at the end of the adhesion assay.
18
GS
specific
activity
50
•
•
•
40
o. 76
z
0
0
0
0
(I)
1.1.1
:t
Q
<
~
20
10
0
15
30
45
TIME (min)
60
0.57
19
Specific activity of glutamine synthetase from cultured
teratoma cells.
The GS specific activity was measured in
cultured teratoma cells after 24 hours of preincubation with
and without actinomycin D, followed by a 1-hour long adhesion assay.
With the exception of one experiment, the
actinomycin D treated cells consistentl,.y showed higher
levels of GS specific activity than the controls.
Based on
10 experiments, the GS specific activity of the control
cells was 0.29 + 0.09 S.E., while that of the actinomycin D
treated cells was 0.38 + 0.12 S.E.
(Table 2).
Using the
values obtained from these experiments, an increase of GS
specific activity of the actinomycin D treated cells over
the control cells was calculated to be 31.4 + 9.9% S.E.
{Table 2).
The exception was experiment 4, in which the.
actinomycin D treated cells showed a decrease in GS specific
activity that was concomitant with a decrease in percent
adhesion.
20
Table 2.
GS specific activity of cultured teratoma cells
preincubated with and without 5
hours.
~g/ml
actinomycin D for 24
For each experiment, an assay for GS specific acti-
vity was· done immediately following an hour long adhesion
assay.
Percent increase in GS specific activity was calcu-
lated as described in the text.
Experiment
Number
Specific Activity
Actinomycin D
Control
Cells
Treated Cells
% Increase in
GS Specific
Activity
1
0.17
0.23
35.3
2
0.27
0.28
3.7
3
0.37
0.41
10.8
4
0.30
0.27
-10.0
5
0.24
0.26
8.3
6
0.38
0.69
81.6
7
0.17
0.27
58.9
8
0.13
0.13
o.o
9
0.57
0.76
33.3
10
0.28
0.53
89.3
Mean +
Standard
Error
0.29 + 0.09
-
0.38 + 0.12
-
31.4 + 9.9%
-
DISCUSSION
A stimulation of GS specific activity by actinomycin D with a concomitant increase in intercellular adhesion
has been observed in cultured mouse teratoma cells.
These
results suggest that intercellular adhesiveness may be enhanced by the actinomycin D stimulated GS activity in cultured mouse teratoma cells.
Various compounds have been found to stimulate GS
activity in eucaryotic cells.
Hydrocortisone (Moscona and
Hubby, 1963; Barnes et al., 1971; Kulka and Cohen, 1973;
Hale, 1977), actinomycin D (Weisman and Ben-Or, 1970;
Rief-Lehrer, 1971; Kulka and Cohen, 1973; Connolly and Oppenheimer, 1975) and an L-glutamine-free medium have all ·
been shown to stimulate GS activity in a variety of eucaryotic cell cultures.
Moscona et al.
(1972) proposed a model
for GS regulation in chick neural retina, in which hydrocortisone stimulates GS activity by inhibiting the product
of a GS repressor gene, allowing the synthesis and accumulation of stable GS mRNA and its subsequent translation
into enzyme.
They also proposed a model for actinomycin D
stimulation of GS activity in this system.
They propose
that actinomycin D binds to the DNA molecule, thus preventing transcription of all gene products, including a labile
suppressor gene.
Stable, preformed GS mRNA is then free
21
22
to translate GS enzyme.
It has been suggested that actinomycin D has a similar effect on other enzymes.
"Superinduction .. of tyrosine
transaminase by actinomycin D has be.en observed in rat
liver (Garren et al., 1964) and in Morris hepatoma cells
(Tomkins et al., 1966).
11
Superinduction 11 refers to the
blocking by actinomycin D of the transcription of a labile
post-transcriptional regulator of tyrosine transaminase
mRNA template translation, thus allowing the translation
of stable tyrosine transaminase mRNA into protein.
Hale (1977) has observed an increase in intercellular adhesiveness when the GS activity in ascites and cultured mouse teratoma cells is stimulated by hydrocortisone.
In the present study, an increased intercellular adhesiveness of cultured mouse teratoma cells was observed when the
GS activity was stimulated by actinomycin D.
The percent
increase in actinomycin D stimulated GS specific activity
in cultured teratoma cells was found to be 31.4 +
9~9%
S.E.
These results are similar to the percent increase of hydrocortisone stimulated GS specific activity in cultured teratoma cells (Connolly and Oppenheimer,
1975) and ascites
.
.
teratoma cells (Hale, 1977), where increases of 38% and 36+
7% S.E. have been observed, respectively.
The percent
increased adhesion by actinomycin D stimulated GS activity
in cultured mouse teratoma cells was found to be 53 + 17%
S.E.
This value is considerably lower than the 669 + 62%
23
S.E. percent increased adhesion reported by Hale (1977) for
hydrocortisone stimulated GS activity.
This difference is
probably due to the fact that Hale (1977) determined the
percent increased adhesion after 24 hours incubation on a
gyratory shaker, whereas, in the present study it was determined after a 1-hour long incubation on a gyratory shaker.
It can be seen from Tables 1 and 2 that both the
percent increase in adhesion and the percent increase in GS
specific activity varied considerably from one experiment
to another.
In addition to slight variations in experi-
mental technique, there are several possible explanations
for this inconsistency.
The actinomycin D solution may have
lost some of its effectiveness due to the repeated thawing
and refreezing during use and storage.
Also, the tissue
culture maintenance process itself tended to select for
those cells that stuck best to the flasks, since the free
floating cells were poured off with the spent medium.
Over
a period of months, this may have also selected for cells
with a higher or lower GS specific activity.
Possibly, the
main factor contributing to the variable results was the
fact that the experiments had not always been done at precisely the same degree of cell confluency.
It has been
shown that as these cells approach confluency, ·that is, as
they form a solid monolayer of cells, the GS specific
activity increases, but declines after confluency has been
reached (Connolly and Oppenheimer, 1975).
If the inter-
24
cellular adhesiveness and the GS activity of the cells did
not increase simultaneously as the cells approached confluency, i.e., if there was a lag period following increased GS activity before an increase in intercellular
adhesion was seen, then one would have expected a difference in the percent increase in each of these characteristics at different degrees of cell confluency.
In one instance (experiment 4) the GS specific
activity of the actinomycin D treated cells was less than
in the control cells.
The reason for the ineffective
stimulation of GS activity by actinomycin D in this experiment is not known, but since a decrease in percent adhesion
was also seen in the actinomycin D treated cells, it appears
that the decreased intercellular adhesiveness of these
cells was related to the decreased GS specific activity.
Oppenheimer et al.
(1969) and Oppenheimer (1973)
have previously shown that mouse teratoma cells require Lglutamine to aggregate.
Also, it has been suggested that
the L-glutamine is required for the synthesis of Dglucosamine-6-phosphate, which is a key intermediate in the
formation of complex cell surface carbohydrates required
for adhesion (Oppenheimer, 1973).
Furthermore, carbohy-
drate containing molecules have been isolated from teratoma
ascites fluid that are capable of attaching to cell surface
receptor sites on ascites teratoma cells and promote an
immediate increase in intercellular adhesion (Oppenheimer
25
and Humphreys, 1971; Oppenheimer, 1975; 1976).
The poor adhesion of tumor cells may be due to
their inability to synthesize, transport, or store Lglutamine (Oppenheimer, 1973).
Since L-glutamine is re-
quired for intercellular adhesion of teratoma cells, the
findings presented here are consistent with the hypothesis
that actinomycin D enhances intercellular adhesion by stimulating GS activity.
The result of this increase in GS acti-
vity is an increase in L-glutamine synthesis, which is
needed for teratoma cell adhesion.
Further investigation
of the regulation of intracellular GS activity may provide
a better understanding of its role in the synthesis of
cell surface molecules involved in intercellular adhesion.
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