CALIFORNIA STATE UNIVERSI_TY, NOR'l:HRIDG,E
HYDHOCOR1riSONE EFFECTS ON ADHESION AND
G LUTAl'IINE SYNTHE'rASE SPEGII•'IC ACTIVITY
DUHING 'J:HE CELL CYCLE OF'.
CULTURED HOUSE TERATDr1A CELIS
11
A thesis submitted in partial satisfaction of the
requir•E.nnents for• th\.':l degx•ee of 1·1asters of Science in
Biology
by
John Charles Scordato
January, 1979
The thesis of' John Charles Scordato is approved:
Cali.fornia State University, No:r•thridge
January, 1979
ii
ACICNOl-JLEDG E!vlENTS
Sincere thanks are extended to Dr. Steven Be
Oppen~
heimer aJ:ld Dr e Narvin H. Cantor for their m.any patient
hours of advice and consul tat ion, laborato1..y facilities,
and for making this thesis possible.
I would also like to
thank Dre Phillip Sheeler• for his assistance and advice in
the Pl"'eparation of' this thesis and his gene:r•ous services as
a member of my thesis committeeo
iii
TABLE OF CONTENTS
ABSTRACT ••• ~ • e
•
•
•
~
~
~
•
$
INfJ.1HODUC':eiON .......... .
•
•
•
•
~
• e • o •
•
•
•
c • c • • • • • •
~
• • • •
• • • • • o • • • • o • e e •
0
•
•
e • • • •
•
•
•
•
•
•
•
•
•
.vi
• • • • o • • s e G
1
8
MATEHIALS AND
NE~THODS.
• o • • • e • • e • • • • • • o • • e G
:reratoma
c~ul tu1~es
............................... ., • • • .. • • 8
~
o
~
• • • •
~
• • • o • • •
Synchronization and dete:t.''.mination of the cell cycle. 8
Adhesior1 assay ........................ .
• • o e • • e e • o •
~
• • •
9
Preparation of enzyme extract ••••••••••••••••••••••• lO
E!rl.zyJ.ne J.\.ssay.
(I.
d
••
a •••••• e.
l1led.ia and ree_gents ..
RES UIJTS • • • • •
e • •
~
e
*
&
•
•
e
0
"
e • • • e e •
0
••
e
(I
••
e e •••••
t&
•
•
G
•
•••
C)
(t
0
• •-
•
••
0
•
••••••
&
•
•
&
".
&
e • o ,. • • o • • • o • •
~
$'
•••
•
11
&
e
fit • • • • •
012
• o
~
e o o • •
o-13
o
'rotal protein per cell during synchrony .............. 13
Intercellular adhesion during the ·cell cycle •••
o~···16
GS specific activity during synchrony in
hydr•ocor·tisone t:r•eated and cont:x~ol cultures
0
(;
~
e
22
GS specific activity vs. adhesion during syncbrony ... 23
Analysis of adhesion in synchronous populati.ona ••••• 23
DISCUSSIONv ~ • • • •.• • • •
.o • • •
o • • • • e • • o • • • • • • • • • ceo o • • • • • e • • • • G32
BIBLIOG. RAPHY., •••••••••••.•••••• " ••• ~ •••••••
iv
e •••••••••••••
38
'rABLES AND FIGURES
'I'ABLE:S
Analysis of adhesion in synchronous populations .... )l
1.
FIGUHES
1.
Gene model for GS I•egulation and induction........ 7
2~
Synchrony of' cell cultures ••••
.3 ~
Pr•otein/cell in synchronomJ cont;rol and
hydrocortisone treated cultures •••••••••••••.••••• l8
Lt.~
IntEn•cellular adhesion in synchronous populations
of control and hydrocortisone treated cultures •••• 21
5..
GS specific nctivit;y during synchrony in control
and hydrocortisone treated cultures .•••••••••••• ~.25
6~
GS specific activity vs. adhesion in
synchronous cultures •••••••••••••••••••••••••••••• 27
7.
b
.....
~
•
., . . . . . . . . . . .
e.15
Analysis of adhesion in synchronous populations
control and hydrocortisone tl. . eated
cultures •••• ~································~··s•30
bet;~H~en
v
ABSrrRACT
HYDROCORTISONE E:F'FEGTS ON ADHESION
Al~D
GLUTANINE
SYN'rHETASE SPECIFIC ACTIVITY DURING THE CELL
CYCLE OF CULTURlill MOUSE TERATOMA CELLS
by
John Charles Scordato
Master of' Science in B1ology
January, 1979
P:Nnrious studies ot: teratoma cells in batch culture
demonstl"Uted that glutamine syntheta:-3e (GS) specific activity 1.nra.s cell density dependent, incl"easing as the culture
approached confluency.
Other studies also showed that in-
tercellula:r• adhes:lon and GS specific activity increased in
the presence of hydrocortisone.
Contluency frequently
fleets a non-cyclic cell populationo
re~·
Hence, in batch cul-
ture the increase in GS activity at confluency may be a
consequence of the cells either leaving the cell cycle or
coming into register at a given point in it, e.g. G1 •
Therefore it is desirable to determine if GS activity and
adhesion are properties of non-cyclicing as well as actively
vi
growing populations.
te:t~:mine
These studies were undertaken to de=
if there is a relationship betvreen intercellular
adhesion and GS specific activity during the cell cycle and
if hydrocortisone efi'ects these parame·ters.
To determine
ii' increased cell adhesiveness and GS specific activity oc-
cur at speci.fic times during the cell cye-le, cell populations we:r•o syrwhronized with thymidine (3m.M) plus colcemid
(. ,Sug/ml) ~
In the synchronized cultures, cellulax• adhe-
siveness and GS specific activity both displayed oscillato:r:y patterns
4-8
l-Ji th
peaks in GS specific activity occurring
hours prior to peal{S in adhesion.
Peaks of adhesive-
ness occur during early interphase and late interphase
(pre~
sumably G /early S and late S/early G accor•ding to the
2
1
mtm~alian cell cycle model of Pardee).
Valleys of a~1e~
siven.ess occur at mid-interphase (p:r•esuraably S) and at cr111
division (mitosis).
Peaks of GS specific activity oGcur
during mid-interphase (S) and near cel·l division ( lat.e
G /H/early G ).
2
1
Adhesion overall is 16.3:!:3.1% highe:r• and
GS specific activity overall is 51.2tl2.3% higher in hydro-
cortisone treated cultures than in untreated cultures
throughout the cell cycle.
Protein/cell in hydrocortil?!one
treated cultures is 20. 2±S. 5% 10\.;er than in untreated cul·~
tures throughout the cell cycle.
These studies demonstrate
that hydrocortisone effects cellular adhesiveness and GS
specific activity during the cell cycle of cultured
toma cells.
vii
tara~
INTRODUCTION
Glutamine serves as a pr•ecuJ•sor for amino acids, proteins1 and nucleotides.
Glutamine also plays an important
role in the fo:t'lnation of complex cax•bohydrates 'Hhich are
necessary for intercellular adhesion
1969;
Oppenheimer, 19'13) ~
(Oppenheimer, et
Evidence suggests that
~1.,
gluta~
mine promotes adhesiveness by donating its 't.amide group in
transe:minating
i'ructose~6-phosphate
to form arrdno sugar con-
taining m.olocules lrhich mediate cellular adbesiono
The
ability of' the cell to promote the px•oduction of glutamine
may be r•eflected in its ability to pr·omote intercellular
adl1esion if there is a relationship bet1r.reen glutamine production and
cellula:~?
adhesiveness o
This study l'tas undei•-
taken to d.cter•m1na if cell adhesion snd glutamine oynthe=
tase act:ivi ty are related during the cell cycle and if hy=
drocortisone, a steroid knm·ln to stimulate glutamine syn=
thetase activity and increase cellular adhesion in logaJ:'ov
ithmic growing cultures of teratoma cells, affects theso
specific par•arneters in synchronous teratoma cultures.
Since glutamine plays such a central role in the metabolism of cells, the regulation of its synthesis is of
special interest and importance.
the
etlZ:fTilB
Glutamine synthetase is
that catalyses the conver•sion of glutamate and
am_rnonia to glutamine {Heister, 1962).
glutamate+ NH
3
+ A':ep ~ glutamine+ ADP +Pi
1
The sa.me enzyme also catalyses the gluta.myltra.ns.ferase l"e""'
action (Lev:l.ntow, ~ ~1., 1955)$
glutamine+ hydroxylamine
~
}(-glutamylhydro.xtunate
Although the second reaction has no
kno~m
.j.-
NH
3
biological signi-
ficance, it is often utilized in the assay for glutruuine
synthetase because tbe product, (\~glutamylhydroxamate, can
be detected colorimetx•ica.lly (a cha.:t..actel'•istj.c brmm color
with the addition of ferric chloride).
The regulation. of glutamine
syntheta~H)
in a "\l'aJ:>ioty of eucaryotic cell typos both
y.i_~:r_,g_.
has been studied
ill
v;i,vo and in
Glutamine synthetase was found to increase g:r•eatly
in grm-ling BeLa cells placed in a medium high in glutamic
acid (20m.M)
ducE:d
(Demars, 1958), and the activity could be rea•
drar~ tic ally
gr•owth medium.
by tho addition of
2mt-~
gluteJnine to the
GS activity has been shm-rn to be dependent
upon the glutarnine concentration of the medium in other cell
types grm,nl i£
ill.£.£
also.
can be effectively reduced by the addition o:r 2.4mM
m:tne to the medium of L-cells previously
lacking glutamine.
L·~cells
The GS activity in mouse
g:t~ovm
gluta~
on medium
If the I.-cells are first grown on
me~
dium supplemented l>rith glutamine (2 .. ~.ml'1), then transferred
to mediurr1 lacking glutamine, an inc:r•ease in GS activity occurs which could be prevented by cycloheximide, indica_ting
that protein
tivity
s~1thesis
is necessary for the increased ac-
(Strunatiadou, 1972).
It has been demonstrated in
va1•ious tissue culture cell types t.hat GS can be induced by
3
various steroid hormones (Bal""nes e~ ~!· 1 1971; Kulka and
Cohen, 1973).
I.f cortisol (alno lmol-m. as hydrocortisone)
ls added to the culture medium o.f mouse £ ... cells, an incl?ease
in GS activity occurs.
If the steroid hormone is deleted
.fl"Om the cultux•e medium afte:p induction, a decl?ease in GS
activity occurs (Ba.l:>:nes et
&·,
1974).
The induction of' glutamine synthetase by hydrocortisone has been extensively studied by Jvloscona and Hubby
(1963) and Hoscona and Piddington (1966 1 1967}, using the
developing chick neural r·etina system.
·'.Phe mechanism o:f
tbe induction in the neural retina is quite complex.
Both
cycloheximide and actinomycin D pr•event induction of GS by
hydrocortisone, protein synthesis being
• " t•J.on 'V-",.oscona.
{H
:tno.uc
Lt
Y.Jeissman and
Ben·~Or,
)
~,.::.•,
r·equir~ed
1968; Hoscona ::;.! al:.
~
f'or trw
1972;
1970; Reif-Lchre:r, 1971).
A model .for the control
or
GS levels and the mode of
induction by hydrocortisone in chick neural retina has been
proposed -vib.ich involves the interaction of a least .five gene
products (Noscona et
regulation of'
GS
Ed·, 1972).
The genes involved in the
according to this model a:t:e:
1) a struc-
tural gene .for GS (codes for messenger-RNA which codes for
the enzyme GS);
2) a repressor gene (codes for a product
which prevents transcription of GS messenger-RNA and desupp:r•essor messenger-ill\!' A);
3) a suppressor gene (codes .for a
product which prevents the translation o.f GS messenger-H.NA
into enzy;.r1e);
L~)
a desuppressor gene (codes t:or a product
which inactivates the suppressor gene, thus allowing G-S
messe:nge:I•-RNA to be translated into enzyme);
5) a degrada-
tion gene (codes for a product which inactivates the enzyme
GS)..
In tb.e nonind.uced (or rep:Pessed) state, only the
I·e~
pressor, suppressor and degradation genes are active, thus
little GS is
activates the
&~A
synthesized~
represso:t~
Hyd:r•ocortisone, the inducer, in-
gene, thus allowing GS
xnessenger~
and desuppressor messenger-RNA to be synthesized.
The
desuppi'€HHW:r.' gene product inactivates the suppressor 9 thus
allm-ring GS messenger-RNA to be tx•anslated into the protein
GS enzyme (Figure 1).
Hs.le {1977) shm..red that the addition of bydr·ocortisone
to the ascites form of teratoma cells and to teratoma tissue
culture cells increases the activity of GS and intercellulal'"
a.dhesiono
Nystrom ( 1978) shm.;recl that actinomycin D also
st:l.m.ulates GS activity and :i.ntf:)rcellular adhesion in cultured
teratoma. cellse
Connolly and Oppenheimer (1975) demonstrated
a cell de:n.sity dependency on GS activity in teratoma cells
and that both hydrocol"'tisone and actinomycin D stimulates GS
activity.
These experiments suggest a complex regulation of
GS a..'l1.d suggest that cell adhesion may be dependent upon the
cell's abillty to synthesize L-glutamine.
The ef'f'ect of cell density suggests that GS activity and
adhesion may relate to the cell cycle.
Con.fluency frequently
reflects a non-cyclic, non-proliferating cell population.
Hen.ce, in batch culture the increase in GS activity at con-
fluency may be a consequence of the cells either leaving the
cell cycle ente:t•ing a quiescent G0 state or coming into reg-
ister at a given point in it, e.g. GJ.
The G0 quiescent
state is believed to be an extension of' the G phase of the
1
cell cycle (Pardee et a~., 1978)$
Theref'ore it is desirable
to dete.Pmi:ne if. GS activity and adhesion are properties of.
noncycling as well as actively gr•owing populations.
6
.!:1£~.-1~
Hypothetical 1'1odel for the Control of Glutarnine
Synthetase Induction in the Embryonic Neural Retina proposed
by Moscona ~t .§:1•
product;
DS:
GS:
(1972}.
repressor;
R:
s tructuro.l gene;
desuppl.,essor gene px•oduct;
suppressor gone product;
DEGR:
De SUPP:
SUPP:
r:
repressor gene
desupp:r•essor gene;
suppressol" gene;
deg:~:~adation
gene;
DG:
S:
de-
gradation gene product.
la)
Noninduced State:
dation genes
a:t~e
The repressor 1 suppr·essor, and clegra'""
aetive e
GS transc:r•iption and ti•anslation
are inhibited, hm·mver some GS templates do SUl"vive and account for the basal levo1 observed. in uninduced conditions.,
lb)
Induced State:
Hydl"'ocortisone induction is achioVEid by
hydx•ocortisone binding to the
its transcription.
repre~H::Ol"
The absence of' the
g.:)ne, thus prevent;ing
repl~essor
gene pi•oduct
f':r•ees the GS structural gene and desuppi"essor gene for tran=
scl.. 1.ption., and the desupp1•essor gene product binds to and
activat;es the suppressor gf;Jne.
in~,.
The result is that th.e GS
structural gene is f'ree for transcription and tl.,a.rlslation
into proteino
lc)
nsuperinduced State: n
Actinomycin D blocks tranrJcrip·-
tion of all genes which control and regulate GS.
However,
any I11J"'qNA that is present which codes for the GS enzyme is
translated into protein.
f
1b
DG
1c
[~.:~~~~ ~u·t~!J
~--Ad
(mRNA)
~
GS
0*--·--
'
MATERIALS .AND HETHODS
~,§lt~_.2*£::l.~E:;;f0S.
strain
RRC~ll+ 1-nn~e
Tissue eultui•es of' mouse 129J ter•atoma
obtained from
Dr~
or
John Lehma.n, Dept.
Pathology, University of Colorado Medical Center (Denver,
Colorado).
Stoc1r cultures viore maintained in 75cm2 tissue
cul tm?e .flasks (Falcon Plastics) in a humidified 5% co 2 atmosphere at .37°Co The g:r:•owth mediu..m. for maintaining cultu:r•es
consisted of: Eagle's minimal essential media (NEM minus
glu~
tru.1th1e) (HHG) supplemented ~d th 10% Fetal bovine serum,
50
microg1"an1s/ml each of
photex~icin
G{~ntamicin
sulf'ate and ?ungizone
B), 200 uni ts/ml penicillin, 200 micrograms/ml
streptornyein sulfate and 2m.H glutamine.
ever;;-
2~
(A:ra~
3 d.ays.
Mediw~1
-vms changed
Cells we:r>e transf'erred weekly using the
mE,dium present to vigorously p:i.pette the cells f::r•om the
f'lasks.
Grovrth rates, cell cycle generation t:i.me, and cell
agg1•ega.tion tvere dete:t>mined using an electl..onic particle
counte~r
{l>1odel 112 LT Celloscope, Particle Data, Elmhur'st,
Illi-nois) t;o count aliquots .from cell suspensions.
Dilu-
tions of the aliquots were made in IEwton (Coulter Electron-
ics, Hialeah, Florida).
Cells were microscopically observed
to detect single cells and aggregates.
§xnct~~at:?-.9n an~~-~.n of ~~~J.l c_y.:s_~
Cells
2
t.rhich reached confluency {app:r•oximately 2 X 105 co1ls/cm )
wez~e tl"'ans.fe:r:•red into 75 cm2 and 150 cm2 f'lasks for the
8
9
pr(~paration
of cell aggregation and glutamine S)'-nthetase
as~
Cells were seeded at a density or ap2
proximately 9 X 103 cells/cm and allm·led to grm·T logar•i thsays l"eapectively.
mically for 2 days in co:rnplate medium ( C!"'J.Elvl) supplemented
with glute..ndne.
SynchroniT.ation of the cells was similar to
the method or Do ida and Olrada ( 1967) except that
deoxycyti~
d:l.ne, lvhich count(n,ac·ts the toxic efi'ects of excess thymid:tne,
t<la£:~
not added..
Cells wero exposed to CM.El-1. (minus
tam:Lne) containing 3mM thymidine fol" 22 hours.
glu~
Cells viere
washed in CN11"M with or Hithout hydr-ocortisone at a concen=
~!::'
tx•ation of 1 X 10 .::>11 f'or 10 hom:'g.
Cells were then exposed
to CHEM containing .5ug/ml of colcemid with or t-vithout
drocortisone ( 10-511) for
4~ 5
hours.
hy~,
Cells entering mitosis
'Here identified microscopically as those cells which round
up
Oil
thG flasks.
Cells
Here
th€Hl vifUJhed in CNBH -vri th or
t<l'i thout hyd:r.•ocortisone (lo=5M).
Cells were then assayed f'or
aggrega:t:i.on and enzyme activity at predetermined time points.
Synch:t:>onization was monitored during the e::tpo:t"'j_ment by noting the mi tot :to index (rounding up of•
cells)~
cell counts
pel" culture, and' amount of proteinJcell ..
Agho~n-~~s~~~ Cells wore pipetted off a 75 cm2 flask using 10 ntl of MMG at
37° Co
Cells ltH:.H''e centrifuged for
5 min-
utes in a conical centrifuge tube at approximately 1000 rpm
in an International Clinical Centrifuge (rotol..
~~221).
The
Nf:IG was decanted and the cells suspended in l ral of IviMG.
10
Using the method
similal~
to that o.f Oppenheimer and Oden-
crantz ( 1972), • 2rnl al1quots -v;ere taken from the suspension,
placed tn 1 dram vials, and incubated at 37°C on a gyx•atory
shaker (11.75 em radius) at 60 rpm 1'or 15 minutes.
A .. 2 ml
aliquot from the suspension was used for the initial counts.
Counts Here deter•udned by diluting the aliquots to 10 ml
'tvith Isoton and quantitatively measu:r•ing the dilutions using
tho electronic particle counter.
Cells were observed on
glass slides photographically using Il.ford HP4 f'ilrn (1/15
sec exposure) to guarantee a single cell suspension before
a:nd aggregates af'ter shaking.
Viability studies using the
dye exclusion test with .1% Trypan Blue showed that viability
r•enHlinE~d
the same before and
aft~n""
shakinge
AcU10sion was
dt'J-
ter·mined as the decrease in single cells into aggregates
over timfj.
fer (pH '7el) as the suspension modium. and centrifuged .for 5
minutes in a conical centrifuge tube at 1000 rpm in an International Clinical Centrif'uge (rotor #221)$
The phosphate
buf'fer was decanted and replaced with 5ml cold phosphate buf-
.fer.
Cells were disrupted in an ice bath by 2-2 minute soni-
cations at a setting of
75
using an Insonator (Hodel N9100,
I.ab-line Instruments Inc., !•Ielrose Park, Illinois, 60160)
equipped with a m:i.crotip.
1 ml of the sonicate was
fr~ozen
11
in liquid N to measure the total cell protein.
Tho remain-
ing sonicate was centrif'uged at 10,000 rpm (12,000 X G) fo!•
30 min at 5°C in a Sorvall RC2-B centz•if'uge (SS-34) rotor.
The supernatant £rom this centrifugation contained the GS
activity.
1~e
extract was stored in liquid N f'or 1-2 days
Cell counts lrere also determined be-
baf'ore it was assayed.
f'ore sonication.
Er~~-~~s~~
Glutrunine synthetase was assayed by the
tamotran8ferase assay (Levi:ntow
Hubby, 1963).
~ ~1.,
glu~
1955; Moscona and
The procedure f'ollowed essentially that of
Moscona and Hubby ( 1963}, with the fo1lo·Hing modifications.
To each reaction tube was added .8 m1 of a solution (pH
5.4)
corwisting o.f 19.4 nli-1 f.fnC1 •H 2 0, 97 ~5 mNNaAc, and ~13 11 glu2
1
tamine. 'I o this vtas added • 7 ml or the enzyme extx•a.ct and
.. 2 rnl of • 59 M adenosine tr:i.phospha te in 0. 01 11 phosphate
buff'er (pH 7.1).
3t3 C.
This mixture was incubated :for 10 rnin at
''.Phen .2 ml of Oell~5 M NH 0H•HCL (hyd:t~o:x.ylamine hydro2
chloride) was a.dded to each tube.
The mixture was incubated
i'or 60 min at 37°C and stopped by addi t:Lon of' 1. 5 ml of' a
solution containing equal volumes of 2. 5N HCL,
chloroacetic acid), and
was
centl~it'uged
f'or
Clinical Centrifuge.
5
5%
Fec1
3
in O.lN HCL.
15% TCA ( tl~i­
The mixtu:t'e
min at 2200 rpm in an International
The absorbence of the supernatant was
read at 500mn using a Beckman Model 2I+ spectrophotometer.
Control tubes containing 0.01 M phosphate buf.fer He:r•e
substituted for the enzyme extr•act.
Glutamylhyd:t,oxamic acid
(GHA) ronnation was determined by comparison with a standard
curve for GHA.
Protein was estimated by the method of Lowry
et a.l. (1951), using bovine serum albumin as a standard.
The specific activity of GS was def'ined as umoles GHA pro=
du.ced per hour per mg protein.
M~g_!_a a~.EiLfl£~
Eagle 1 s autoclavable minimal essential
medium minus glutamine containing Earle's salta and Heat In ...
activated Fetal Bovine Serum were obtained from GIBCO (Grand
Isla.nd, New York).
Thymidine, hydroxylamine hydrochloride,
bovine serum albumin (f'raction V), glutamylhydroxamic acidp
gentamycin r:m.lfate
jJ
and
hydrocortisone~21-phosphate
wer·e
ts.ined from Sigma Chemical Co. (St. Louis, Iviisr.wuri L
o~d.ne
tr•iphoBphate,
L~glutamine,
ob~
Aden=
a.nd colce:mid -v;ere obtained
from Calbiochem (Los Angelos, Calif'.).
FUngizone Has ob=
tained from E. M. Squibb & Sons (New York, N.Y.).
§.lpch~.<?..~il"
of
ce11_~}3~
In synchronous control and hy-
drocortisone treated cultures having 28 hour cell cycle
times, the .fi:t"'st division occuri•e<?- within 2 hours after the
release from colcemid treatment and
incr·ease in cell nurn.bera).
\<laS
very acute (85-110%
The second division was not as
sharp, occurring betHeen hours 28. and 30 after the release
from colce.mid and culminating by hour 32.
cultures with 20 hour cell cycle times, the
In synchronous
~irst
division
was delayed betvreen 6 to 10 hours after the release f'rom col-
cemid in hydrocortisone treated cultures.
Di v·ision in con-
trol cultures of 20 hour cell cycle times occurred at
hours after the l"elease from colcemid treatment.
14
The second
division occurred 20 hours after the f'irst division and division was not as shax•p as the first indicating that by the
time the second division occurs, the cultures begin to depart
from s:YJachrony (Figure 2).
appreciable difference in total px•otein and soluble protein
content in these studies, ei thex.. total protein/cell or solu-
ble protein/cell can be used as additional evidence t:or the
occur1..ence of' cell division.
The protein/cell data is cor-
robora.tive of the timing of cell divis:ton determined by cell
nmubers.
The protein/cell in hydrocortisone treated cultures
13
fl.gl,'£.,6 _2..:..
Synchrony of' cell cultures.
At 2 hour• time
points, after the release from colcemid, cells l<Jere l'"emoved
2
f'rom 75 cm f'lasks usi.ng a neoprene policeman and suspended
in MHCL · Cell counts were obtained using an elect.I•onic particle counte1•.
Graph is demonstrative of a typical experi-
ment f'or control or hydrocortisone treated cultures.
Graph
was obtained f:r•om a hydrocortisone treated experiment in
which the cell cycle time was 20 hours.
Synchronization of:
cells is described in Materials and 1Y1ethods.
15
8
7
6
'd'
5
0
.....
X
N
E
v
........
4
~
-'
-..3
w
3
v
2
1
0
-14 -12 -10
~a
-s -4
o 2
-2
I
4
6
a
10 12 14 16 18 20 22 24 26
HOURS I\FTER DIVISION
MITOSIS
tiTOS!S
16
is
20.2±5.5%
cell cycle.
less than in control cultures throughout the
Immediately f'ollovring division, the total pro-
tein/cell and soluble protein/cell is slightly rn.o.r>o than
T~~
half' the protein/cell just prior to division.
content
of' protein/cell increased as the cell proceed-ed ::<:r•ough the
cell cycle until cell division occurred again (i•':i.gm.:"e 3).
,!!lte,!"cellular adhesiqn d!J}"i_pg the__£_~,1.1
c;r,~le::.
Intercellu-
lar adhesiveness displays oscillatory patterns during the
cell cycle.
In a typical experiment, both control and hy-
drocortisone treated cultures, mitosis is always a point
v.rhere adhesiveness is low.
In cultures of' cell cycle times
of' 28 hours, aill1esion is low during and just
vision.
a~ter
cell di-
Adhesion rises and remains high 4-12 hours af'ter
cell division in hydrocortisone treated cultures.
In con-
trol cultures, adhesion remains lm.v until hours 8-12 with a
peak in adhesione
At hour
14,
adhesion begins to drop in
both control and hydrocortisone treated cultures.
At hour
16, aill1esion is at a low point with control and hydrocortisone treated cultures having nearly the se.me adhesiveness.
By hour 20, adhesion rises again but the adhesion peak is
not as high as earlier in the cell cycle.
hesion drops again leveling
period
or
orr
at hour
24.
At hour 22 adDuring this
the cell cycle, the hydrocortisone treated cul-
-ture remains slightly higher than control cultures.
At
hours 26 and 28 (mitosis and dell division} adhesion is
17
Figl!]-"e ;..
Protein/cell
cortisone tt•eated cul tux
~lynchronous
control and hydro.,
Cell density \·ms obtai:i:1ed from
For protci.n/cell, at 2 hour time points,
2
cells "t<rere removed f'rom a 15'0 cm .flask and suspended in
Figur·e 2 (.D).
0. OlN phosphate bu.f.fer (pH 7.1).
Cell counts were
using an electronic particle counter.
determined by the method of Lcn--rry
suspensions used for the GS assay.
obtalnt~d
Protein content was
~ ~.
( 1952) .from protein
Control (0) and hydro-
cortisone treated cultures (.A) having cell cycle times oJ' 20
hours were examined.
18
3.0
2.6
8
1
2.2
6
"J;:
~
'¢
....X
0
·e~
5
'1f:J
0-1
C\l
E
.,...
1.8
4
n
M
!Jll
_,
,...
,...
....X
o,
-A
w
u
rn
z
........
3
2
1.4
1
1.0
0
-14 -12 -10-8 -6 -4 -2
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28
HOURS AFTER DIVISION
1MITOSIS
1,,\ITOSIS
""
19
again at a letAJ point only to rise again after cell division.
Hydrocortisone treated cultur·es remain more adhesive than
control cultures after cell division (Figure
4).
All experiments shmrT oscillatory patterns of adhesiveness.
However, peaks and valleys o:f adhesiveness do not
match for each time point :from experiment to experiment
due, possibly, to variability between populations.
Parallel
control and hydrocortisone treated cultures were examined
simultaneously providing an internal control :for every experiment$
Peaks or valleys of adhesiveness·were within 2 hours
o:f each other in such parallel cultures.
When adhesion ln
control cultures is higher than in hydrocortisone treated
cultures, it is due to differences in the occurence of peaks
and valleys.,
In general, hydrocortisone treated cultures
demonstrate higher corresponding peaks of adhesion and exhibit more ad.hesiveness during valleys of adhesion than control cultures.
Using the
% increased_
%mean
fo1~ula:
hydrocortisone
adhesion
adhesion -
%mean control
adhesion
% mean control
adhesion
X 100
adhesion overall in hydr>ocortisone treated cultures is 16.)
±3.1% higher than in control cultures.
Although no determination of the S phase was madeg and
i.f teratoma cells behave as other mammalian cells, then the
S phase occurs for 1/3 of the cell cycle during its mid ... portion ( Pa1~dee
~ ~·,
1978).
The data \vould then imply that
20
f~&ure
4.
Intercellular adhesion in synchronous populations
of control and
hyd1~ocortisone
treated cultures.
cont1•ol (0) and hydrocortisone treated
(~)
Parallel
cultures. were ex-
amined simultaneously having cell cycle times of 28 hours.
At 2 hour time points, cells tvere removed from a 75 cm2
.flask and suspended in r·'IT1G.
Cells were rotated f'or
15 min
using the adhesion assay described in Materials and Methods.
Using an electronic particle counter, adhesion was measured
as the decrease in single cells after rotation.
50
40
V1l
...D
....1
LU
u
....,
w
0
z
v;
LL.
0
w
t,""!l
ct
I.Y
~
u
w
Q
~
10
0-2
b~
0
2
4
\
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34
HOURS AFTER DIVISION
MITOS!S
'
MITOSIS
.,;.·
--
~.
---- -------
~--
22
adhesion is highest in G1 /early S in both cultures. There
is a valley in mid-interphase (presumably S). Late S/early
G cells exhibit a small rise in adhesion and a valley oc2
curs again during mitosis and cell division.
Qf!~$!_fi~!!:Y.~t;z: 9-ur.fng_~J'1!C~- in_hydrocor~is~~
~~li.n..£~
C5_Ytl'Grol....£.111 tures.
GS specif'ic ac ti vi ty in both
control and hydrocortisone treated cultures exhibit oscillatory
pattet~s
during the cell cycle.
From 2 hydrocorti-
sone and 2 control experiments having 20 hour cell cycle
times, there are usually 2 peaks ot' GS specit'ic activity
during the cell cycle.
In a hydrocortisone experiment,
peaks of GS specit'ic activity occurred at -6, 0, 10, 18,
and
24
hours after the
f'i1~st
specific acti.vit;y occurred at
division while valleys of GS
-4, 4
to 8, 16, and 20 hours.
In a control experiment, GS specific activity peaks
occur~
red at -12, -2, 6, and 16 hours. after the first division
't-¥hile valleys of' GS specific activity occurred at
2, and 12 hours.
~10
to -8,
Acco!•ding to the model of the cell cycle
developed by Pai•dee (1978), one peak of GS
~pacific
activ-
ity would occur during early to middle S while the second
peak occurs during late G2 to early G1 • In comparison o:f
corr•esponding peaks ·and valleys of' GS specific a.ctivi ty between control and hydrocortisone treated cultures, the peaks
in hydrocortisone treated cultures are higher than peaks in
control cultures while the valleys found in hydrocortisone
--
~
----
~-
23
treated cultures have been .found to be as low, but navel..,
lower, than in control cultures {Figure
5).
Using the i'or•-
mula:
GS speci.fic
%mean GS specific
act. \fith hydro- act. in control
% increased GS cortisone treatment
cultures
X 100
specific activity% mean GS speci.fic act.
in control cultures
%mean
GS ~pecif'ic
activity overall in hydrocortisone t:r•eated cul-
tures is 51.2 12.3% higher than in control cultures.
GS spec.,!fic f!.ctivity vs. adE~~-i?n du~;!:!!B~][l1Cl'l,;.l:_'OTIJ...:;.
of GS specif'ic activity occur
4-8
Peaks
hours bef'o:r•e peaks of' ad=
hesion in both control a.11d hydroco:t•t:isone treated cultures.
GS spocific activity peaks usually oceur du.rir:<g valleys in
adhesion 't.vhile valleys of' GS specific activity usually occur
during peaks of' adhesion (Figure 6).
does not lend themselves to statistical analysis because or
variability between individual populations.
internally controlled.
Experiments are
Certain trends do develop, however,
in both control rund hydrocortisone treated cultures.
Based
on the mean value of adhesion and standard deviation r.or each
expel~iment,
at, 0, or; is assigned for each time point,
The assig:mn.ent of a +, 0, or·-, is a basis on ho-u-1 much of an
incl'ease OI" decrease takes place as pea.ks and valleys occur
Fig~a2!.
GS specific activity during s7nchrony in control
and hydrocortisone treated cultures.
Corresponding peaks
and valleys are compared betHeen control {0) and hydrocort;j_ ...
sone treated (A) cultures having cell cycle tDnes of 20
hours.
At 2 hour
cm2 f'lasks.
t~1e
points, cells were removed from 150
GS specific activity \-Jas assayed as described
in Materials and Nethods.
Ar•r•otvs indicate
where mitosis oc-
curs.
----~~-~--
2.2
2.0
1.8
1.6
C\1
'o
....
X
....
u
4
\.J
1.4
1.2
w
a.
41)
vi
1.0
C)
.8
.6
.4
-14 -12 -10 -8 -6 -4 -2
1
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30
HOURS AfTER DIVISION
MITOSIS
·
·
1
PAilOSIS
26
figure 6!.
GS speci.fic activity vs .. adhesion in synchronous
cultures.
At 2 hour time points, .from a hydrocortisone
treated culture having a 20 hour cell cycle time, GS
.fie activity {A) is compared to adhesion {0).
sults occur .for control cultures as well.
points where mitosis occurred.
speci~
Similar re-
Arrows indicate
27
2.4
60
2.2
2.0
50
1.8
1.6
40
1.4
z
-
1.2
30
1.0
.8
20
.6
10
-6 -4 -2
t
.4
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28
HOURS AFTER DIVISION
MITOSIS
tiTOSIS
p
•
28
during the cell cycle..
An increase or decline in the amount
or 1/2 the standard deviation or less from 2 consecutive
time points would render the second time point the same value as the first.
An
increase in the amount of the standard
deviation from a -point would result in a 0 value at the
second point.
A +value would have resulted
at the second
point if the increase was 2X the amount ot: the standard deviation.
Control values at hours 12, 16, and 24, are higher than
hydrocortisone treated values.
However, within 2 hours of
these time points, the corresponding peaks or valleys in hydrocortisone treated cultures exhibit more adhesiveness than
control cultures (Figure 7 and Table 1).
29
Eigure 7.
Analysis or adhesion in synchronous populations
bet1-reen contl"ol and hydl'•ocortisone treated cultures.
F~rom
cultures having 28 hour cell cycle times, analysis is based
on+, 0, -,to demonstrate the trend which develops throughout the cell cycle.
From
4
dir£erent experiments run simul-
taneously betHeen control (0) and hydrocortisone treated
(A) cultures, the comparison is observed for each experiment and the sum of+, 0, -,at each time point is calculated from the four experiments.
30
-2 0
\
2
4
MITOSIS
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36
HOUftS AFTER DIVISION
\
MITOSIS
31
Table 1.
Peaks and valleys of adhesiveness in contr•ol and
hydrocortisone treated cultures based on+, O, and
ment from four parallel experiments.
~
assign-
The sum at each time
point is calculated from the four experiments.
Hours
Aftel'•
Control
Cultures
Cell
Division
Hydrocortisone # of Times HC AdheTreated
sion is Higher than
Cultures
Control Adhesion Subtract Number of Times
Control Adhesion is
Higher than HC Adhesion
-2
0
2
1
0
-2
-2
0
2
0
1
0
4
-1
1
3
6
-1
2
g
0
3
3
3
10
12
2
2
1
-1
2
-2
14.
-3
0
3
16
18
2
1
0
2
0
0
0
3
1
3
3
0
1
-1
-4
-3
-3
0
2
-1
32
-2
1
4
4
4
1
1
J4,
0
0
0
20
22
21+
26
28
30
DISCUSSION
An increase in intercellular adhesion associated with
hydrocortisone-stimulated levels
or
GS specific activity
during the cell cycle in synchronized cultures or teratoma
cells has been observ·ed.
The evidence presented here indi-
cates that adhesion and GS speciric activity during the cell
cycle are oscillatory with peaks
or
GS specific activity oc-
curring just prior to peaks or adhesiveness.
Peaks
or
ad-
hesiveness can be enhanced in the presence of hydrocol"'tisone.
The evidence also suggests that hydrocortisone may possibly
enhance teratoma cell adhesion in culture during the cell
cycle by stimulating cellular GS activity levels.
Hydrocortisone has been used extensively to induce GS
activity in the chick neural retina (Piddington a..1"ld Moscona,
1967; Kulka and Cohen, 197)).
Connolly and Oppenheimer
(1975) demonstrated that cultured mouse teratoma GS activity
is subject to stimulation by hydrocortisone.
Moscona has
p:r•oposed that hydroco:t,tisone acts in the chick neural r•etina
by inhibiting transcription of the GS repressor gene, alloHing the synthesis and accumulation of stable GS messengerRNA and its subsequent translation into the enzyme.
Immuno-
chemical studies have shown that the increase in GS activity
during hydrocortisone induction is due to enzyme synthesis
. o:f the GS enzyme
1972}.
(Hoscona
~ §.1·,
1968; Hoscona et !i1·,
Connolly and Oppenheimer (1975) using cycloheximide
32
3.-')\·
synchronized mouse leukemic L5178Y cells demonstrated that
glycosyltranaferase activity is highest during the S phase
of the cell cycle and that little glycosyltransf'erase activity is observed during mitosis.
Shur ru1d Roth (1975) have
postulated that in cell cultures, a transformed cell or cell
at mitosis can
glyco~ylate
cis-glycosylation.
itself.
Thus a cell performs
The mechanism involved for transformed
cells or cells at mitosis is that a cell under these conditions is able to bind their surface acceptors with their own
surface glycosyltransferases, rather than bind acceptors on
adjacent cells.
If this is occurring in teratoma cultures,
it would account for the low adhesiveness at mitosis.
Hm.J-
everj low ad..hesiveness du:Ping interphase in both control and
hydrocortisone treated cultures suggests that the mechanism
o:f ad.hesion :must be more complex than just dependent upon
glycosyltransferase activity during the cell cycle.
Metabolism is also important in intercellular•
adhesion~
It has been previously shown that mouse teratoma cells require L-glutrunine for adhesion.
Evidence presented suggests
that teratoma cells require L-glutamine for the synthesis of
D-glucosamine-6-phosphate, a key intermediate in the formation of cell surface complex carbohydrates required for adhesion (Oppenheimer, 1973).
Pouyss'egur, lrJillingham and
Pastan (1977) using Balb/c 3T3 cells, AD6 mutants, showed
that adhesion is pr•evented when glycoprotein synthesis does
not occur but can be restored when an intermediate metabolite
34
(5ug/ml) or actinomycin D {5ug/ml} indicate that this system
may also be regulated at the gene level.
The oscillatory pattern or GS activity during the cell
cycle observed in both control and hydrocortisone treated
cultures suggest that the enzyme may be regulated by fluctuations in degradation of the enzyme or by oscillatory repression.
Oscillatory repression is accomplished when an
enzyme product, usually a metabolite of the system, represses
the synthesis of that
enZYL~e,
and involves oscillations in
the concentrations of this product, mRNA,· and enzyme protein
(}1itcheson, 1971).
In this case, the oscillations which oc-
cur for glutamine synthetase may be due to the levels of
glycolipids, glycoproteins, or metabolites such as nucleotide-sugars.?
or glucosamine-6-phosphate, intermediates in
the synthosi.s o:f glycolipids and glycoproteins.
I.f the glu-
tami.ne concentration fluctuates during the cell cycle, then
glutf'.mine may act as the regulatory molecule that is responsible for the oscillatory patterns observed.
Oscillations
could also be the I•esult of changes i.n the quaternary structure of the enzyme.
Roseman (1970} has postulated that cell sur.race glycosyltransferases may be involved in intercellular adhesion by
trans-glycosylation of a terminal sugar of' a glycoprotein or
glycolipid.
The glycosyl transferase from one cell uses the
.glycoprotein or glycolipid fz•om an adjacent cell as a substrate for the cell surface enzyme.
Bosmann (1974} using
35
is added past the point of: inhibition in the glycoprotein
synthesis pathway.
I.f N-acetlyglucosrunine-6-phosphate, the
first intermediate past the block from glycosrunine-6 ... p
~
N-acetylglucosa.mine-6-P, is added to these mutant cells glycoprotein synthesis occurs and adhesion is restored.
In
Balb/c 3T3 cells, AD6 mutants, it is the defect in the enzyme responsible for the acetylation or
glucosamine~6-P
be-
cause feeding of N-acetylglucosamine-6-P to these cells restores adhesion.
Hydrocortisone not only increases levels o.f GS specific
activity, but also influences lysosomal activity as well as
plasma membrane activity (at high steroid concentrations).
Lewis, Symons, and Ancill (1970) have demonstrated that bydrocortisone stabilizes lysosomal membranes at concentrations of 10- 6 to 10-~.. By examining the release o:f acid
phosphatase and,B-glucur•onidase activity from lysosomes, it
was found that the activity and release or these enzymes is
inhibited at steroid concentrations o:f 10- 6 to 10-4!1.
Therefo1..e, at these concentrations, ste:r•oids become inc or·~
porated into the lysosomal membrane, stabilizing the membrane and preventing the release of lysosomal
enz~nes.
At
steroid concentrations o:f 10-3 to 10-~ lysis of lysosomes
occurs releasing the enzymes packaged in them.
The concen-
tration of' hydrocortisone-21-phosphate in these cell cycle
.experiments was 10-5M.
The stabilization of' lysosomal mem-
branes with hydrocortisone at a concentration o:f 10-5M
---
--·--·-------
~.
---~--
--------
resulting in a decrease in lysosomal enzyme activity may ef ..
feet the plasma membrane by decreasing degradative glycoprotein and glycolipid enzyme activity present.
Tpis may stim-
ulate adhesion.
It has been shown that the chemical compositions of rat
liver• lysosomal membranes and the plasma membrane are similar (Thines-Sempoux, 1967).
It has also been shown that
steroids interact with artificial membranes suggesting that
the action of steroids on biological membranes may result
directly from their interaction with lipid, independent of
other membrane components (Bangham et g., 1965).
This may
also be the situation with the interaction of 10-5M hydl"ocortisone-21-phosphate and the plasma membrane of cultured
teratoma cells.
In these present experiments, it has been den1onstrated
that adhesion throughout the cell cycle is influenced by the
presence o.f hydrocortisone.
Hydrocortisone increases adhe-
sion possibly by increasing levels of GS specific activity
thus permitting
manufactu1~e
of' more L-glutamine f'or the pro-
duction of complex carbohydrates required for adhesion.
If'
the production of complex carbohydrates is increased, this
would allow for more substrate present, for example, for
cell surface glycosyltransferase activity.
The production
of sugB.r-nucleotides may also increase and allovl for additional substrate.
The increase in adhesion in the presence
of' hydrocortisone may also involve the incorporation of
37
hydrocortisone into the plasma membrane promoting stabilization that increases intercellular adhesiveness.
The :ln-
crease in adhesion_ may be also due to the stabilization or
lysosomes by hydrocortisone which diminishes the release of
lysosomal enzymes that may reduce protease activity and/or
degradation of cell surface glycoproteins and glycolipids.
Although the incorporation of hydrocortisone into the plasma membrane and lysosomal membrane has not been investigated here, they can not be ruled out as possible explanations for the increase in adhesion by hydrocortisone
throughout the cell cycle.
BIBT..,IOGRAPHY
Bangham, A.D .. , Standish, M.}'I., and vJeissmann, G. {1965).
The Action oi' Steroids and Streptolysin S on the
Permeability of' Phospholipid Structures to Calcium.
:I2Er!l_al ~.2f_l!ole~~Q_logy 13, 253.
Barnes, P.R., Youngberg, D., and Kites, P.Ag (1971).
Factors ~ffecting the Production of Glutrunine in
Cultured Neuse Cells. Q~.ll. ,Ph;ysiology 7.J.., 135.
Barnes, P.R., Hersh, R.T., and Kitos, P.A. (1974).
Regulation of Glutarnine Synthetase in L Cells by
Cortisol and L-G lut amine. _fP.;. Vi tr2_..,2, 230.
(1974). Cell Plasma Membrane F~ternal
Glycosyl transf'erases: Acti.vi ty in the Cell
Mitotic Cycle. B:i.ocb.imica et Bi,.£PJ:ysica Acta 339, 438.
Bosmann, H.R.
Sur~.race
Cofl..no lly, D. i' ~ and Oppenheimer, S. B. ( 1975) • Cell Dens i tyc•
Dependent Stimulation of Glutamine Synthetase Activity
in Cultur"ed .House Teratoma Cells. !!_~p~rirnental Cell
B~se~rcJL9~,
460.
Del.J:ars,. R. (1958). The Inhibition o.f Glutamyl 1l'ransferase
Fo:r•mation in Gult;u:ees of' Human Cells. Biochilaica e c
Biop~~£a.~c~~z,
435.
Dolda, Y. and Okada, S.
(1967). Synchronization o.f L5178Y
Cells by Successive Treatment with Excess Thymidine
and Colcemid. §eperi~.e_nta.l_.9_ell__Besear•ch M-8, .540.
Hale, R.A.
(1977)e A thesis entitled "Hydrocortisone
Induced Stimulation of Teratoma Cell Adhesiveness 11 ~
Kulka, R.G. and Cohen, H.
(1973). Regulation of Glutamine
Synthetase Activity of: Hepoma Tissue Culture Cells by
Glutamine and Dexamethasone. ~£~~~~910~~~~1
~,6738.
Levintov,r, L., Heister, A.. , Hogeboom, G.H. and Kuff~ E.L.
(1955) Studies on the Relationship Between the
Enz~natic Synthesis of: Glutamine and the Glut~ayl
T:.r•ansferase Reaction. Journal of the. American Chemical
~21
530L}.e
....-
38
-"----
.,.~,'
-
-----~-----
---~---
39
Lewist D.A., Symons, A.M., and Ancill, R.J. {1970).
Stabilization-lysis Action of Anti-inflawmatory
Steroids on Lysosomes. Journal of' Pharmacy end
Pba:t'nl&.£,?1.£By 22, 908.
---
The
Lowry, 0. H., Rosenbrough, M. J., l?arr, L.A., and Randall, Re
J. (1951). Protein Measurement with the Folin Phenol
Reagent. Journal of Biol~ical Ch~~ist~y 191, 265
Mitchison, ,J.M.
(1971). Enzyme Synthesis. The Blolog;y of'
the Cell Cycle pp. 159-180. Cambridge Unived~'-'·- Q~
Press, -craiTI.br1 dge •
Moscona, A.A. and Hubby, J.L.
(1963). Experimentally
Induced Changes in Glut~~otr&~sferase Activity in
Embryonic Tissue. QeveloEm~qt~l Biolog.._!._l, 192.
Hoscona, A.A. and Piddington, R. (1966}. Stimulation by
Hydrocortisone of Premature Changes in the Developmental
Pattern of Glutamine Synthetase in Embryonic Retina.
,2i..o.2.fii!.n1s~a et Biophysica Acta 121, 409.
1-foscona, A.A., Noscona, M.H. andSaenz, N. (1968). Enzyme
Induction in the Embryonic Retina: The Role of
Transcription and Translation. Proceedings of the
£~~dem;t:: ofv §Eien~J u~~1;-~.lb0:"
.
N., Frenkel, N., Noscona, A. A. ( 1972). Regulatory
11echanisms in the Induction oi' Glutamine Synthetase in
h'mbryonic Retina: I.rn...ilunochemical Studies.
~~ntal Biologx ?~, 229.
?-1osconaf
Nystrom. R.R. (1978). A thesis entitled "Actinomycin D
Stimulation of Teratoma Cell Adhesion."
Oppenheimer, S.B. (1973).. Utilization oi' It-Glutamine in
Intercellular Adhesion: Ascites Tumor and Embryonic
Cells. ~pe!:,imental Cell Resear~l!_ll, 175.
Oppenheime.n•, S.B., Edidin, M., Orr, C.H., Roseman, s.
(1969). An I.-Glutamine Requirement for Inte:x·(~ellular
Adhesion. Proceedi~f' the National AcademL-2.£
§ci~n~es, .u~, 13~
Oppenheimer, S.B. and Odencrantz, J.
(1972). A Quantitative
Assay i'or Heasuring Cell Agglutination: Agglutination
of Sea Urchin Enbryo and Mouse Teratoma Cells by
Oonconavalin A. Ex~epimental Cell Ren~C:.£1 7:1, 479.
-
~.
-~--------~
-----
Pardee, A.B., Dubrow, R., Ha.rnlim, J.L., and Kletzien, R.F.
(1978).
Animal Cell Cycle. Annual Review of
--=~
Biochemistr•y bt-7., 715.
Piddington, R. and ~'ioscona, A.A.
(1967}.
Precocious
Induction of' Retinal Glutamine Synthetase by
Hydrocortisone in the Embryo and in Culture:
Differences iri Tissue Response.
~..2!?.~ ~_9_a_ Ac t~Jlt1,
Biochimi2a
Age
~~
429.
Reif-Lehrer, L.
( 1971). Actinomycin-D enhancement of
Glutamine Synthetase Activity in Chick Embr·yo Retinas
Cultured in the Presence of Cortisol. Journal of'
~];_J~i2.;I;£EI-.2l, 30 3.
Pouyss 1 egur, Jq itlillingha:m, N., and Pastan, I.
(1977).
Role of Cell Surface Carbohydrates and Proteins in
Cell Behavior: Studies on the Biochemical Reversion
of an N-Acety1glucosarnine-Deficient Fibr•oblast Mutant.
~~oceedinf.!:S
of the :Nati9!:!-.af. Apademv of Scie~,
t::l{-3~
Roseman, So (1970). The Synthesis of' Complex Carbohydrates
by Nultiglycosyltransf'erase Systems and their Potential
F'unction in Intercellular Adhesion.
!~J~~s
of
Lipi~,
C~!:l~ a~£
270.
Shur~ B .. D.
and Roth, S.
(197_5). Cell Sl:trface Glycosyltransferases. ~~-et ~;i_o_EhY.~a Ac~a Y,.~, lt73~
Stamatiadou, M.N.
(1972)~
Effects of Glutamine on the Two
Catalytic Activities of Glutamine Synthetase in
Cultured Mouse Cells Strain L. Biochemical and
B~o..EhW:..cal HeseaJ:ch -~E:.~cat_?.~
Thi~1es-Sempoux, D.
(1967). Chemical Similarities BetV-reen
the Lysosome and Plasma Hembrane. ~;i.:E_<?~~ln_ical Jo2:;11~
192_, 20P.
\velssman, H. and Ben-o'r~, S.
(1970).
Regulation of' Glutamine
Synthetase in the Em.bryonic Neural Retina in Organ
Culture: Biochemical a:g~q_ 112.2-~al Research Oommunicat~on~~