CALIFORNIA STATE UNIVERSI'rY, NORTHRIDGE
CELL DENSITY DEPENDENT STIMULATION OF
GLUTAMINE SYNTHETASE ACTIVITY IN
CULTURED MOUSE TERATOMA CELLS
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
Daniel Thomas Connolly
Nay, 197.5
The thesis or Daniel T. Connolly is approved:
Calirornia State
Univer~ity,
May, 1975
ii
Northridge
F
ACKNOWLEDGEMENTS
Sincere thanks are extended to Dr. Steven B.
Oppenheimer for his many patient hours of advice and
consultation, laboratory facilities, and for making
this thesis possible.
I would also like to thank
Dr. Marvin Cantor and Dr. Charles Spotts for their
help in the preparation of this thesis and for their
generous services as members of my thesis committee.
iii
p
TABLE OF CONTENTS
INTRODUCTION. • • • • • •
• • • • • • • • • • • ,1
MATERIALS AND METHODS • • • • • • • • • • •
Teratoma cultures. • • • • • • • • •
Ascites grown teratoma • •
• • 7
• • • 7
• • • • • • • • 7
Preparation of enzyme extract. • • • • •
Enzyme assay • • • • • • • • • • •
Media and reagents • • • • • • • •
RESULTS • • • • • • • • • • • • • • • •
Cell cultures. • • • • • • • • • •
• 8
• • • • • 9
• • • • .10
• • • • .12
• • • • .12
Cell density and glutamine synthetase. • • .12
Glutamine synthetase from ascites grown
cells.
• • • •
.. •
• • • • • • • • • • • .18
Effect of dbcAMP, cycloheximide,
actinomycin D, and hydrocortisone.
• • • .19
DISCUSSION. • • • • • • • • • • • • • • .. • • • .23
BIBLIOGRAPHY .. • • • • • • • • • • • • • • • • • .30
iv
LIST OF TABLES AND FIGURES
TABLES
1
EFFECT OF dbcAMP, CYCLOHEXIMIDE,
ACTINO~£CIN
D AND HYDROCORTISONE
ON GS SPECIFIC ACTIVITY• • • • • • • • • • 20
FIGURES
1
GROWTH OF TERATOMA CELL IN CULTURE • • • • 13
2
CHANGES IN GS SPECIFIC ACTIVITY
WITH TIME IN CULTURE • • •
v
• •
•
.•••• 15
p
ABSTRACT
CELL DENSITY DEPENDENT STIMULATION
OF GLUTAMINE SYNTHETASE ACTIVITY
IN CULTURED MOUSE TERATOMA CELLS
by
Danie.l Thomas Connolly
Master of Science in Biology
May, 1975
A density dependent stimulation of glutamine synthetase activity (GS) has been observed in cultures of
mouse teratoma cells.
GS specific activity increases as
cultures approach confluency to a level greater than twofold over the basal level found in sparse cultures.
After confluency the GS specific activity returns to the
basal level found in sparse cultures.
The stimulation
could not be attributed to age of cultures, medium or
glutamine depletion, cell leakage of GS, or change in the
amount of cellular protein.
Dibutyryl cyclic-AMP plus
theophyline repressed GS activity both in cultured teratoma and in teratoma obtained from ascites grown tumors •
.
The evidence suggests that the stimulation is contact
mediated.
The stimulation is prevented by cycloheximide
vi
and is therefore influenced by de
protein.
~
synthesis of
Increased activity is observed upon addition
of hydrocortisone or actinomycin D.
The results are
discussed with respect to the possible role of GS in
controlling cellular adhesiveness.
vii
p
INTRODUC 1riON
Glutamine in cells serves as a specific precursor
for proteins, amino acids, and nucleotides.
In mouse
teratoma cells it has been shown that glutamine is required for the formation of complex carbohydrates which
are apparently involved in intercellular adhesions
(Oppenheimer, et al, 1969; Oppenheimer, 1973).
Ascites
grown teratoma cells required the addition of exogenous
glutamine to promote cellular aggregation.
Evidence
suggested that glutamine promotes adhesiveness by its
role in transaminating fructose-6-phosphate to form
amino sugar containing molecules which mediate cellular
adhesion (Oppenheimer, et al., 1969; Oppenheimer, 1973).
The present study was undertaken to determine if teratoma cells contain the enzyme glutamine synthetase
essential in forming L-glutamine required for cell adhesion in this system.
/
Since glutamine plays such a central role in the
metabolism of cells, the regulation of its synthesis is
of special interest.
To date, only one pathway for the
synthesis of glutamine is known, although other possible
pathways have been investigated and cannot be conclusively eliminated (Meister, 1962).
1
Glutamine synthetase
p
2
(GS) is the enzyme that catalyses the conversion or
glutamate and ammonia to glutamine (Meister, 1962).
Glutamate + NHJ + ATP
Glutamine + ADP + P.
1.
The same enzyme has also been shown to catalyse the
glutamyltransferase reaction (Levintow, et al., 1955).
Glutamine + hydroxylamine ----7 y-glutamylhydroxamate +
NH
3
Although the biological significance of this second
reaction is not known, it is often utilized in the
detection or GS because the product,
V -glutamylhydrox-
amate, has a characteristic brown color with the addition
or ferric chloride (Meister, 1962).
GS (L-glutamate ammonia ligase ((EC 6.3.1.2)) ) has
been isolated from a variety or sources including the
bacterium Escherichia coli (Shapiro, et al., 1968) and
--
--
Bacillus subtilis (Deuel, et al., 1970), sheep brain
(Meister, 1962), pea and rat liver (Tate and Meister,
1971), and chick neural retina (Sarkar, et al., 1972).
These enzymes all show certain similarities in amino acid
composition and molecular weight, but are not identical
physically or chemically.
Subunit molecular weights vary
,
3
%
from 42,000 in neural retina GS (Sarkar, et al., 1972)
to 50,000 in bacterial GS
(Shapiro,~
al., 1968; Deuel,
et al., 1970).
The regulation of GS has been studied in various
eucaryotic cell types both in vivo and in vitro.
Demars
(1958) found that GS specific activity could be greatly
increased by growing HeLa cells on a medium high in
glutamic acid (20 mM) and that the activity could be
drastically reduced by the addition of 2mM glutamine to
the growth medium.
GS activity has been shown to be
dependent upon the glutamine concentration of the medium
in other cell types grown in vitro also.
Stamatiadou
(1972) found that GS activity could be effectively
reduced by the addition of 2.4mM glutamine to the medium
of mouse L-cells previously grown on medium lacking
glutamine.
If the L-cells were first grown on medium
supplemented with glutamine
(2.~I),
then transferred to
medium lacking glutamine, an increase in GS activity
occurred which could be prevented by cycloheximide,
indicating that protein synthesis was necessary for the
increased activity.
Similar results have been obtained
with cultured Chinese hamster cells (Tiemeier
and
Milman, 1972), and with cultured hepatoma cells (Kulka
and Cohen, 1973).
It has also been shown in various
tissue culture cell types that GS can be induced by
4
various steroid hormones (Barnes, et al., 1971; Kulka
and Cohen, 1973).
The induction of glutamine synthetase has been
extensively studied in the developing chick neural
retina.
GS activity begins to rise dramatically on the
seventeenth day of incubation of the embryo (Rudnick and
Waelsch, 1955; Moscona and Hubby, 1962), a change which
corresponds with the functional maturation of the eye
(Wald and Zussman, 1938).
The increase can be brought
about prematurely by the addition of hydrocortisone to
organ cultures of the developing retina (Piddington and
Moscona, 1967; Moscona and Piddington, 1966).
The mechanism of the induction of GS by hydrocortisone in the neural retina has been well studied and is
quite complex.
Since both cycloheximide and actinomycin
D can prevent induction of GS by hydrocortisone, protein
synthesis is required for the induction (Moscona, et al.,
1972; Moscona, et al., 1968; Weissman and Ben-Or, 1970;
Reif-Lehrer, 1971).
A
mo~el
for the control of GS levels and the mode
of induction by hydrocortisone in the chick neural retina
has been proposed which involves the interaction of at
least five gene products (Moscona, et al., 1972). According to this model the genes involved in the regulation
of GS are: 1) a structural gene for GS (codes for
5
messenger-RNA which codes for the enzyme GS); 2) a
repressor gene (codes for a product which prevents the
transcription of GS messenger-RNA and desuppressor
messenger-RNA); 3) a suppressor gene (codes for a product
which prevents the translation of GS messenger-RNA into
enzyme);
4)
a desuppressor gene (codes for a product
which inactivates the suppressor gene, thus allowing GS
messenger-RNA to be translated into enzyme);
5)
a degrad-
ation gene (codes for a product which inactivates the
enzyme GS).
Under uninduced conditions, only the repres-
sor, suppressor and degradation genes are active, thus
little GS is synthesized.
Hydrocortisone, the inducer,
inactivates the repressor gene, thus allowing GS
messenger-RNA and desuppressor messenger-RNA to be synthesized.
The desuppressor gene product inactivates the
suppressor, thus allowing GS messenger RNA to be translated into protein.
The induction of GS in chick neural retina has been
shown to be dependent upon specific cell interactions
(Morris and Moscona, 1970; 1971).
If neural retinas were
grown intact in organ culture, they were responsive to
GS induction by hydrocortisone.
If however, the retinas
were dissociated and the cells grown in monolayer, GS
could not be induced by hydrocortisone.
Cells which were
dissociated but allowed to immediately form aggregates
6
were intermediate in their response to hydrocortisone.
These experiments implied that multicellular, histotypic
organization was necessary ror the induction of GS.
The present study rocuses upon levels or GS in
monolayer culture or mouse teratoma or various cell densities.
Since teratoma is a cell type derived rrom germ
cells, it has a rairly high potential ror dirrerentiation
(Pierce, 1974), and in this way may be similar to the
developing chick neural retina.
It has been found that
a cell density dependent stimulation or GS speciric
activity accompanies the approach or confluency or the
cultures, and that the stimulation is not dependent upon
the age or the culture or upon medium or glutamine
depletion.
The stimulation can be prevented by the
addition or cycloheximide and thererore appears to be
dependent upon protein synthesis.
GS speciric activity
can be increased by addition or hydrocortisone phosphate
(1o- 6M) or actinomycin D (5 Mg/ml) to the medium and can
be decreased by the addition of glutamine or dibutyryl
cyclic-AMP/ (dbcAMP) plus theophylline to the culture
medium.
MATERIALS AND METHODS
Teratoma cultures.
Cultures of mouse teratoma strain
RRC-14 were obtained from Dr. Michael Edidin of The
Johns Hopkins University.
Stock cultures were maintained
on 15 mm plastic tissue culture dishes or 75 cm2 tissue
culture flasks (Falcon Plastics) in a humidified 5% C0
atmosphere at 37°C.
2
The growth medium consisted of
Eagle's minimal essential medium supplemented with 10%
fetal bovine serum, 125 units/ml each of penicillin and
streptomycin, and 2mM L-glutamine.
every 3-4 days.
Medium was changed
Cells were transferred weekly using 0.2%
trypsin in pH 7.4 HEPES (O.OlM) buffered Hanks' balanced
salts solution (HH) to remove the cells from the flasks.
Cell number and growth rates were determined using an
electronic particle counter (Model 112 LT Celloscope,
Particle Data, Elmhurst, Ill.) to count aliquots taken
from the trypsinized single cell suspensions.
Dilutions
were made/in Isoton (Coulter Electronics, Hialeah, Fla.).
Cells were observed microscopically to insure that single
cells, not aggregates, were present.
Ascites grown teratoma.
Teratoma cells, originally ob-
tained in 1966 from Dr. Leroy Stevens of the Jacksop
7
8
Laboratory, were maintained by intraperitoneal passage
in young male 129/J mice, as described previously
(Oppenheimer, et al., 1969).
develop for
5-15
The cells were allowed to
days until the animals began to show
abdominal bloating.
The animals were sacrificed by cer-
vical dislocation and the peritoneal contents emptied
into 10-15 ml cold HH.
The cells were washed twice in
cold HH by low speed centrifugation in an International
clinical centrifuge (rotor #221), and either resuspended
immediately in phosphate buffer for sonication, or incubated for one hour in
50
ml HH containing 1.0 mM dibuty-
ryl cyclic AMP plus 1.0 mM theophyline or containing
1.0 mM 5'-AMP.
After the incubation, the cells were
collected by centrifugation, resuspended in phosphate
buffer, and sonicated.
Preparation of enzyme extract.
Cells were seeded onto'
tissue culture flasks at initial concentrations of 5Xl05
cells/flask (low density seeding) or 4X10 6 cells/flask
(high dens-ity seeding).
Medium was changed every 3-4
days and 24 hours before the cells were to be removed for
the GS assay.
L-glute~ine
Medium containing all components minus
(MMG) was substituted for complete medium in
some of the cultures.
added to MMG.
Supplements, where indicated,
~ere
Enough flasks were taken for the GS assay
9
to give a total cell count, when pooled, of approximately
1 o8 cells.
To collect the cells, the medium was removed,
replaced with cold HH, the cells scraped from the flasks
with~ineoprene policeman, and centrifuged for
5
min in
15 ml conical centrifuge tubes at 2/3 speed in an International clinical centrifuge (rotor #221).
The HH was
decanted and replaced with 3-6 ml cold phosphate buffer
(O.OlM, pH 7.1).
Cells were disrupted in an ice bath by
three 30 sec sonications at a setting of 1.5 using an
Insonator (Ultrasound Systems Inc., Farmingdale, New York)
equipped with a microtip.
at 12,000XG for 30 min at
fuge (SS-34 rotor).
The sonicate was centrifuged
5°0
in a Sorvall RC2-B centri-
The supernatant from this centri-
fugation contained the GS activity.
The extract was
always assayed on the same day that it was prepared.
It
was found that it could be stored frozen at -12°0 for one
week with little loss of GS activity, but after 10 days ,
much activity was lost.
E'nzyme assaY.
Glutamine synthetase was assayed by the
glutamotransferase assay (Levintow, et al., 1955; Moscona
--
and Hubby, 1963).
The procedure followed essentially that
of Moscona and Hubby (1963), with the following modifications.
To each reaction tube was added 0.8 ml of a.
Solution (pH 5.4) containing 10 ~moles each of NaH Po
2 4
10
and MnC1 , 180 ~moles L-glutamine, and 100 ~moles sodium
2
acetate. To this was added 0.7 ml of enzyme extract
(0.3-0.7 mg protein) and 0.2 ml of 0.5mM adenosine triphosphate in O.OlM phosphate buffer (pH 7.1).
ture was incubated for 10 min at 37°C.
This mix-
Then 0.2 ml of
O.l5mM hydroxylamine hydrochloride was added to each tube.
The mixture was incubated for 60 min at 37°C, and the
reaction was stopped by addition of 1.5 ml of a solution
containing equal parts of 2.5N HCl, 15% trichloroacetic
acid, and
5%
ferric chloride in O.lN HCl.
The mixture
was centrifuged for
5 min at top speed in an International
clinical centrifuge.
The optical density of the super-
natant was read at 500nm using a Bausch and Lomb Spectronic 20 colorimeter.
Control tubes containing distilled
water substituted for hydroxylamine were used as blanks
for each extract.
Duplicate experimental tubes were
prepared for all assays.
Glutamylhydroxamic acid (GHA).
formation was determined by comparison with a standard
curve for GHA.
al.,
--
Lowry et
Protein was estimated by the method of
(1951) ... using bovine albumin as a standard.
The specific activity of GS was defined as
~moles
GHA
produced per hour per mg protein.
Media and Reagents.
Eagle's minimal essential medium
containing Earle's salts, and Hanks' balanced salts
-.
11
solution were obtained from GIBCO (Grand Island, New
York).
Fetal bovine serum and penicillin-streptomycin
concentrate (25,000 units/ml) were obtained from
Microbiological Associates (Los Angeles, CA).
Difco
1:250 trypsin was obtained from Difco Laboratories
(Detroit, MI).
L-glutamine, hydroxylamine hydrochloride,
bovine albumin (fraction V), glutamylhydroxamic acid,
21
cycloheximide, actinomycin D, w6 ,o -dibutyryl adenosine3':51-cyclic monophosphoric acid (dbcAMP), and hydrocortisone-21-phosphate were obtained from Sigma Chemical
Co. (St. Louis, MO).
Adenosine triphosphate, and HEPES
buffer (N-2-hydroxyethylpiperazine-N'-ethanesulfonic adid)
were obtained from Calbiochem (Los Angeles, CA).
r
t
RESULTS
Cell cultures.
Under the conditions or this study,
teratoma cells grew in a monolayer with an average doubling time or
24
hours.
When cells were seeded at low
initial density (5Xl05 cells/rlask) they reached a saturation density or 1.6Xl05 cells/cm2 • When seeded at high
initial density (4Xlo 6 cells/rlask) a saturation density
2
or 2Xl05 cells/cm could be attained (Fig. 1). Microscopic observation conrirmed that at saturation density,
virtually all cells on the rlask were in contact with one
another.
Under conditions of low density seeding, con-
rluency was reached after 6 days in culture, while under
conditions of high density seeding conrluency was reached
after only 3 days in culture.
Cell density
an~
glutamine synthetase.
Cultures were
removed at various times arter seeding and assayed for
GS activity.
It was found that the speciric activity of
GS was dependent upon the density of the cultures.
In
sparse cultures, GS activity levels were low, but as the
density or the cultures increased, the GS level increased,
eventually reaching a peak which was approximately 2 rold
higher than the basal level (Fig. 2).
12
Peak GS activity
r
13
'
Figure 1.
Growth of teratoma cells in culture.
Cells were seeded onto f'lasks at an initial density of
5Xl05 cells/flask ( 6 ) or 4X10 6 cells/flask ( 0 ) .
To
count the cells, flasks were removed and the cells not
attached to the flask were poured off and counted.
Attached cells were removed from the flask using 0.2%
trypsin and counted.
An electronic particle counter was
used to count the cells.
The cell number reflects the
sum of attached and unattached cells.
r
14
I
-'o
1.0
1.0
~
C\J
-·~.
~
(f)
._j
:._j
0.1
w
u
3
6
DAYS
9
r
15
I
Figure 2.
Changes in GS specific activity with time
in culture.
Cells were seeded onto flasks in complete
medium at initial concentrations of 4X10 6 cells/flask
(1, 2) or 5Xl05 cells/flask (3,
24
4).
Medium was replaced
hours before cells were to be removed for the GS assay
with either complete medium (1, 3) or MMG (2,
4).
GS
activity was assayed as described in Materials and
Methods.
Bars represent standard deviations of the means
of 2 to 6 separate assays.
~
I .
16
I
>-
1.5
1-
>
F
u
<(
J.O
4
u.
-LL
-0
2
w 0.5
(L
3
.(f)
3
6
·DAYS
9
17
was reached on the sixth day of low density seeded
cultures, and at the third day of high density seeded
cultures.
The third and sixth days represent the times
at which the respective cultures reached confluency
(Fig. 1).
If cells are removed from flasks at these
times by trypsinization or by scraping, they come off in
sheets or in aggregates which are quite resistant to
dissociation.
these cells.
This indicates intimate interaction between
In post confluent cultures, GS
specific
activity began to fail and eventually dropped to a level
approximating the level found in sparse cultures (Fig. 2).
Peak GS activity appeared at different times in high
and low density seeded cultures; therefore the increase
in activity cannot be related to the age of the cultures.
To check if the increase in GS activity was due to medium
depletion or conditioning, medium was replaced with either
complete medium or MMG for all cultures 24 hours before
each assay.
The increase in GS activity appeared in the
presence or absence of glutamine, suggesting that
glutamine/depletion is probably not responsible for the
activity increase.
GS activity was significantly reduced,
however, in cultures in which 2mM glutamine was present
in the growth medium.
In an experiment to further demon-
strate that glutamine depletion was not causing the increase in GS, 2Xl0 6 cell/flask were seeded in MMG and
18
allowed to grow for 3 days. At this time the cell density
was o.6Xl05 cells/cm2 • It should be noted that the
cultures would have reached confluency by this time had
glutamine been present (Fig. 1).
If glutamine depletion
was responsible for the increase, it might be expected
that these low density cultures would exhibit high GS
activity.
It was found, however, that the GS activity
resembled activity from low density cultures in which
glutamine had been removed only 24 hours before the assay.
Since the kinetics of glutamine utilization have not been
studied in these cells, however, the idea that glutamine
concentration may play a role in the increase of GS
activity cannot be totally excluded at this time.
GS activity was not detectable in the medium of
sparse cultures, suggesting that low specific activity in
sparse cultures is not due to cell leakage.
To insure
that the increase in specific activity was not due to a
decrease in total protein, the amount of protein per cell
was determined for different aged cultures.
For one day
old high density seeded cultures (low GS) there was
?.22Xl0-7mg protein/cell and in three day old high density
seeded cultures {high GS) there was ?.50X10-7mg protein/
cell.
Glutamine syntpetase from ascites grown cells.
Teratoma
19
cells grown intraperitoneally in 129/J mice were found
to contain GS with a specific activity comparable to the
basal level found in sparse cultures of teratoma.
The
mean specific activity of eight separate assays was 0.32
(Standard deviation=O.lO).
The cells grown under these
conditions are found in single cell suspension in the
ascites fluid, and do not readily form aggregates.
The
ascites form of the tumor is no longer in the embryoid
body state (Oppenheimer, et al., 1969).
Effect of dbcAMP, cycloheximide,
hydrocortisone.
actino~ycin
D, and
The addition of lmM dbcAMP plus lmM
theophylline to high density, logarithmically growing
cultures of teratoma cells
24
hours before the assay
reduced GS specific activity markedly.
The specific
activity found in these cultures was low in comparison
with the level found in the control cultures seeded at,
the same density (Table 1, Column C), but similar to the
level observed in low density cultures (Table 1,
Column D)./
When ascites grown teratoma cells were incubated for
one hour with lmM dbcAMP plus lmM theophylline, specific
activity was reduced in
4
separate assays by 26% when
compared with control cultures.
no effect on GS activity.
Addition of
5 1 -AMP had
Addition of dbcAMP to cell free
20
TABLE 1.
Effect of dbcAMP, cycloheximide, actinomycin D and
hydrocortisone on GS specific activity.
The supplements
were added in the concentrations indicated to the medium
(MMG) of cultured teratoma cells
assay.
24
hours before each
Specific activity (umoles GHA formed/hour/mg
protein) was measured in cultures to which supplement was
added (column B), and in control cultures to which no
supplement was added (column A).
The values given are
the mean specific activities of 2 assays in the case of
dbcAMP and of 3 assays in the other experiments.
are given in parentheses.
Ranges
The ratios given in column C
are obtained from the quotient B/A.
The ratios given in
column D are obtained from the quotient B/0.65.
The
number 0.65 corresponds to the specific activity observed
in low density cultures (see Figure 2, graph 2, day 1).
21
TABLE 1
Supplement
GS Specific
Cell
Density
Activity
at Time
of
Addition
(cells/
2
em )
Minus
Plus
Supple- Supplement
ment
A
B
-
lmM dbcAMP
+
1.5Xl05
(dense)
lmM Theophylline
Cycloheximide
(5ug/ml)
0.96
(0.921.09
0.61
(0.470.69
Ratio of' Sp.
Act. of Supplemented
Cultures to
Unsupplemented
Cultures
Same
Cell
Density
Low
Cell
Density
c
D
0.64
0.94
0.79
0.86'
0.71
!(0.71(sparse)
0.74)
0.56
(0.56)
o.6Xlo5
0.71
(sparse) (0.710 .. 74)
1.01
(0.991 .. 08)
1.41
1.55
Hydrocortisone 0.6Xl05
0.98
0 .. 71
(sparse) (0.71(0.97(10- 6M)
1.00)
0.74)
1.37
1.51
Actinomycin D
(5ug/m1)
5
o.6x1o
22
extracts did not reduce enzyme activity, suggesting that
dbcru~P
does not act by direct interaction with GS.
In experiments using cycloheximide (5p.g/ml), actinomycin D (5mg/ml) and hydrocortisone (10-~), the supplements were added to the medium of relatively sparse,
logarithmically growing teratoma cells, 24 hours before
the assay.
GS specific activity in cultures to which
cycloheximide was added showed lower FG specific activity
than control cultures of the same density (Table 1.,
Column
c.).
The specific activity of these cycloheximide
supplemented cultures was also slightly lower than the
level observed in low density, non-supplemented cultures
(Table 1., Column D.).
Actinomycin D and hydrocortisone
phosphate increased GS specific activity in comparison
with control cultures of the same cell density (Table 1,
Column
c.).
DISCUSSION
A stimulation of glutamine synthetase activity has
been observed in cultures of mouse teratoma cells.
The
evidence presented suggests that the increased GS
specific activity was dependent upon cell density and
possibly cell contact.
GS activity rose as the cultures
approached confluency, reached a peak when saturation
density was attained, and fell again to the basal level
in post-confluent cultures.
Experiments were performed
to show that the stimulation of GS activity was probably
not attributable to the age of the culture, medium depletion or conditioning, glutamine depletion, cell leakage, or changes in the cellular protein concentration.
Although protein synthesis seemed to be involved in the
activity increase, it has not yet been determined if the
increase in GS activity was due to an increased rate of
GS synthesis, a decreased rate of GS degradation, or a
decreased/rate of GS inhibitor synthesis.
Preliminary experiments were performed using two
inhibitors of protein synthesis to determine more clearly
the nature of the GS stimulation.
Cycloheximide, an
inhibitor which blocks protein synthesis at the translational level was tested at a concentration of
23
5
~g/ml.
The inhibitor was added to cultures in which GS was increasing from a basal level, to an elevated level
24
hours later (compare day 1 and day 2 of Fig. 2).
Cyclo-
heximide was found to prevent any increase in activity
over the
24
hour incubation period.
GS levels in the
cycloheximide supplemented cultures remained approximately at the level found in preconfluent cultures, while
the level in control cultures to which no cycloheximide
was added showed increased GS activity. over the
incubation period (Table 1).
24
hour
These data imply that pro-
tein synthesis was required for the increase in GS
activity to occur.
The cycloheximide data presented thus far still
leaves some interesting but as yet unanswered questions.
First of all, does cycloheximide, at the concentration
tested, completely block protein synthesis?
Is the
effect of cycloheximide on GS activity due to the inhibition of the synthesis of new GS?
2-5 ~g/ml
In chick neural retina,
cycloheximide prevented the incorporation of
90-lOO% of labeled amino acid, and prevented the induction of GS by hydrocortisone (Moscona, et al., 1968).
It
seems likely then, that 5 ~g/ml cycloheximide added to
teratoma cultures would be sufficient to inhibit protein
synthesis as well.
In reference to the second question,
a sensitive assay for the detection of newly synthesized
enzyme is not available at this time.
It is therefore
not possible to state conclusively that the increased GS
specific activity is due to newly synthesized enzyme, nor
that cycloheximide prevents the increase by inhibiting
the synthesis of new GS.
It should be noted, however,
that even though other explanations cannot be ruled out,
synthesis of new GS is the most likely explanation for
the increased GS activity observed in confluent teratoma
cultures.
Actinomycin D, an inhibitor of protein synthesis at
the transcriptional level, was also added at a concentration of
5
wg/ml to preconfluent cultures.
In this
instance an increase in GS activity was observed over the
controls.
As with the cycloheximide data, drawing de-
finitive conclusions without further experimentation to
determine the extent of inhibition of RNA and protein
synthesis, concentration effects, and specifically therate of GS synthesis, is impossible.
In other systems,
however, actinomycin D has also been observed to cause
11
superindliction 11 of some enzymes, suggesting that similar
mechanisms may be involved.
11
Superinduction 11 of tyrosine
transaminase by actinomycin D has been observed in rat
liver (Garren, et al., 1964) and in Morris hepatoma cells
(Tomkins, et al., 1966).
"Superinduction" of GS by
actinomycin D has been observed in chick neural retina
T
26
{Weisman and Ben-Or, 1970; Rief-Lehrer, 1971) and in
cultured hepatoma cells (Kulka and Cohen, 1973).
The "superinduction'' of tyrosine transaminase in the
presence of actinomycin D has been explained on the basis
that actinomycin D prevents the transcription of a labile
post-transcriptional regulator of tyrosine transaminase
template translation, thus allowing the translation of
stable tyrosine transaminase messenger-RNA into protein
{Tomkins, et al., 1966).
Weissman and Ben-Or (1970) have
proposed that a similar mechanism may be involved in the
11
superinduction 11 of GS in the presence of actinomycin D.
Moscona has also proposed the presence of a labile posttranscriptional inhibitor of GS translation based upon
other experimental evidence (Moscona, et al., 1968;
Moscona, et al., 1972; Jones and Moscona, 1974).
The
actinomycin D data presented here is not inconsistent
with this hypothesis.
The stimulation of GS by hydrocortisone has been
commonly observed (Piddington and Moscona, 1967; Barnes,
et al., 1971; Kulka and Cohen, 197 3).
Data is presented
here which shows that GS in mouse teratoma cultures is
also subject to stimulation by hydrocortisone.
To pre-
confluent cultures, 10- 6M hydrocortisone phosphate was
added 24 hours before the assay.
These cultures showed
increased GS activity over controls.
Moscona has pro-
27
posed that in the chick neural retina, hydrocortisone
acts by inhibiting the product of a GS repressor gene,
thus allowing the synthesis and accumulation of stable GS
messenger-RNA, and its subsequent translation into GS
(Moscona, et al., 1968; Moscona, et al., 1972).
Further
experimentation would be necessary to determine if a
similar mechanism is involved in GS stimulation by hydrocortisone in mouse teratoma cultures.
The addition of
lmM dbcAMP plus lmM theophylline to preconfluent teratoma
cultures prevented the increase in GS activity observed
in controls, and the GS level was reduced to a level found
in low density cultures or in post-confluent cultures.
In other experiments, ascites grown teratoma cells showed
decreased GS activity after 1 hour incubation with lmM
dbcAMP plus theophylline.
This data is consistent with
the proposed hypothesis that increased GS activity is a
density dependent phenomenon.
cAMP is known to control, a
diversity of cellular activities, including levels of
specific enzymes (Pastan and Perlman, 1970).
Further, it
is well known that in logarithmically growing cultures,
cAMP levels are lower than in stationary cultures, that
cAMP levels increase when cultures reach confluency, and
that the changes in cAMP levels seem to be contact dependent (Seifert and Paul, 1972; Bannai and Sheppard,
1974.).
The prevention of the increase of GS in pre-
,
28
confluent cultures by dbcAMP may suggest that cAMP
mediates the drop in GS activity in post-confluent
cultures.
According to this hypothesis, in preconfluent
cultures of teratoma cAMP levels would be low, allowing
an increase in GS activity as the cells approach confluency.
When the cells reach confluency, the amount of
intracellular c.AJ.'.iP would rise, preventing further synthesis of GS and bringing about the decrease in GS
specific activity observed in post-confluent cultures.
Since L-glutamine is an important intermediate in
numerous metabolic pathways, including the formation of
complex carbohydrates present on the cell surface
(Oppenheimer, et al., 1969; Oppenheimer, 1973;
Oppenheimer, 19?5), cellular GS activity may play a role
in phenomena such as contact inhibition of growth and
intercellular adhesion.
It has been observed that hamster
fibroblasts and transformed hamster fibroblasts taken
from sparse cultures are markedly less adhesive than cells
taken from confluent cultures
(Edwards,~
al., 19?1).
It is possible that the increased adhesiveness in the
latter cells is related to an increase in GS in more
dense cultures.
The adhesive properties of teratoma cells
in culture also change with cell density.
The percentage
of cells unattached to the flask drops as GS levels increase and aggregates from confluent cultures are signifi-
29
cantly more resistant to dissociation than aggregates
from sparse cultures (Connolly and Oppenheimer, unpublished).
GS levels in teratoma cells taken from ascites
tumors of 129/J mice are relatively low.
These cells are
observed now to grow as single cells and do not readily
form aggregates.
Further study may elucidate more clear-
ly the relation of GS levels in mouse teratoma cells to
intercellular adhesion.
,
30
BIBLIOGRAPHY
Bannai, S. and Sheppard, J.R. (1974).
and Cell Contact. Nature 250 62.
Cyclic AMP, ATP
Barnes, P.R., Youngberg, D. and Kitos, P.A. (1971).
Factors Affecting the Production of Glutamine in
Cultured Mouse Cells. Cell Physiology 7], 135.
De Mars, R. (1958). The Inhibition of Glutamyl Transferase Formation in Cultures of Human Cells. Biochimica et Biophysica Acta 27, 435.
Deuel, T.F., Ginsburg, A., Yeh, J., Shelton, E.G. and
Stadtman, E.R. (1970). Bacillus subtilis Glutamine
Synthetase. Journal of Biological Chemistry 245,
5195.
Edwards, J.G., Cambell, J.A. and Williams, J.F. (1971).
Transformation by Polyoma Virus Affects Adhesion of
Fibroblasts. Nature New Biology 231, 147.
Garren, L.D., Howell, R.R., Tomkins, G.M. and Crocco, R.M.
(1964). A Paradoxical Effect of Actinomycin D: The
Mechanism of Regulation of Enzyme Synthesis by Hydrocortisone. Proceedings of the National Academy of
Sciences, U.S.A. 52, 1121.
Jones, R.E. and Moscona, A.A. ( 1974). Effects of Cytosine
Arabinoside on Differential Gene Expression in Embryonic Neural Retina. Journal of Cell Biology 61,
688.
Kulka, R.G. and Cohen, H. (1973). Regulation of Glutamine
Synthetase Activity of Hepatoma Tissue Culture Cells
by Glutamine and Dexamethasone. Journal of Biological Chemistry 248, 6738.
~,
31
Levintow, L., Meister, A., Hogeboom, G.H. and Kuff, E.L.
(1955). Studies on the Relationship Between the
Enzymatic Synthesis of Glutamine and the Glutamyl
Transferase Reaction. Journal of the American
Chemical Society 77, 5304.Lowry, O.H., Rosenbrough, N.J., Farr, L.A. and Randall,
R.J. Protein Measurement with the Folin Phenol
Reagent. Journal of Biological Chemistry 193, 265.
Meister, A. ( 1962). Glutamine Synthetis. In The Enzymes
(P.D. Boyer, H. Lardy and K. Myrback, Eds.)
pp. 443-468. Academic Press, New York.
Morris, J.E. and Moscona, A.A. (1970). Induction of
Glutamine Synthetase in Embryonic Retina; Its
Dependence on Cell Interactions. Science 167, 1736.
Morris, J.E. and Moscona, A.A. (1971). The Induction of
Glutamine Synthetase in Cell Aggregates of Embryonic
Neural Retina: Correlations with Differentation and
Multicellular Organization. Developmental Biology
~~
420.
Moscona, A.A. and Hubby, J.L. (1963). Experimentally
Induced Changes in Glutamotransferase Activity in
Embryonic Tissue. Developmental Biology 7, 192.
Moscona, A.A., Moscona, M.H. and Saenz, N. (1968).
Enzyme Induction in the Embryonic Retina: The Role
of Transcription and Translation. Proceedings of
the National Academy of Sciences, U.S.A. 61, 160.
Moscona, A.A. and Piddington, R. (1966). Stimulation by
Hydrocortisone of Premature Changes in the Developmental Pattern of Glutamine Synthetase in Embryonic
Retina. Biochimica et Biophysica Acta 121, 409.
Moscona, M., Frenkel, N., and Moscona, A.A. (1972). Regulatory Mechanisms in theinduction of Glutamine Synthetase in Embryonic Retina: Immunochemical Studies. Developmental Biology 28~, 229.
'i
32
Oppenheimer, S.B. (1973). Utilization of L-glutamine in
Intercellular Adhesion: Ascites Tumor and Embryonic
Cells. Experimental Cell Research 77, 175.
Oppenheimer, S.B. (1975). Functional Involvement of
Specific Carbohydrate in Teratoma Cell Adhesion
Factor. Experimental Cell Research, in press.
Pastan, I. and Perlman, R. (1970). Cyclic Adenosine
Monophosphate in Bacteria. Science 169, 339.
Piddington, R. and Moscona, A.A. (1967). Precocious Induction of Retinal Glutamine Synthetase by Hydrocortisone in the Embryo and in Culture: Age Dependent
Differences in Tissue Response. Biochimica et Biophysica Acta 141, 429.
Pierce, B.G. (1974). The Benign Cells of Malignant
Tumors. In Develo mental As ects of Carcino enisis
and Immunity King, T.J., Ed •• Academic Press, Inc.
New York.
Reif-Lehrer, L. {1971). Actinomycin-D enhancement of
Glutamine Synthetase Activity in Chick Embryo Retinas
Cultured in the Presence of Cortisol. Journal of
Cell Biology 51, 303.
Rudnick, D. and Waelsch, H. (1955). Development of Glutamotransferase and Glutamine Synthetase in the Nervous
System of the Chick. Journal of Experimental Zoology
128' 309.
Sarkar, P;"!c., Fischman, D.A., Goldwasser, E., and Moscona,
A.A. (1972). Isolation and Characterization of ·
Glutamine Synthetase from Chicken Neural Retina.
Journal of Biological Chemistry 247, 7743.
Sarkar, P.K. and Moscona, A.A. (1973). Glutamine Synthetase Induction in Embryonic Neural Retina: Immunechemical Identification of Polysomes Involved in Enzyme Synthesis. Proceedings of the National Academyof Sciences U.S.A. 70, 1667.
"i
33
Schwartz, R.J. (1973). Control of Glutamine Synthetase
in the Embryonic Chick Neural Retina: A Caution in
the Use of Actinomycin D. Journal of Biological
Chemistry 248, 6426.
Seifert, w. and Paul, D. (1972). Levels of Cyclic AMP in
Sparse and Dense Cultures of Growing and Quiescent
3T3 Cells. Nature New Biology 240, 281.
Shapiro, B.M. and Ginsburg, A. (1968). Effects of Specific Divalent Cations on Some Physical and Chemical
Properties of Glutamine Synthetase from Escherichia
Coli. Biochemistry 7, 2153.
Stamatiadou, M.N. (1972). Effects of Glutamine on the
Two Catalytic Activities of Glutamine Synthetase
in Cultured Mouse Cells Strain L. Biochemical and
Biophysical Research Communications 47, 485.
Tate,
s.s., and Meister, A. (1971). Regulation of Rat
Liver Glutamine Synthetase. Proceedings of the
National Academy of Sciences, USA 68, 781.
Tiemeier, S.C. and Milman, G. (1972). Regulation of
Glutamine Synthetase in Cultured Chinese Hamster
Cells: Induction and Repression by Glutamine.
Journal of Biological Chemistry 247, 5722.
Tomkins, G.M., Thomson, E.B., Hayashi, s. Gelcherter,
T.D., Granner, D.K., and Pederdofsky, J. {1966).
Tyrosine Transaminase Induction in Mammalian Cells
in Tissue Culture. Cold S~ring Harbor Symposia on
Quantitative Biology 31, 3 9.
Wald, G. and Zussman, H. (1938). Carotenoids of the
Chicken Retina. Journal of Biological Chemistry 122,
499-460.
Weissman, H. and Ben-Or, s. {1970). Regulation of Glutamine Synthetase in the Embryonic Neural Retina in
Organ Culture. Biochemical and Biophysical ResearchCommunications 41, 260.