Vol. 8 1, No. 3, 1978 April 14,1978
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 832-840
SUPERINDUCTION OF ORNITHINE DECARBOXYLASE BY ACTINOMYCIN D AND CORDYCEPIN Max Costa Institute of Materials Science and Department of Laboratory Medicine University of Connecticu't Storrs, Connecticut 06268
Received
December 19,1977
Cordycepin and Actinomycin D when added 2.0 - 3.5 hrs. following the addition of serum or serum and dibutyryl CAMP to quiescent cultures of Chinese hamster ovary cells, resulted in "superinduction" of ornithine decarboxylase at 4 hrs. If these agents were added from 0 to 2 hrs after stimulation, ornithine decarboxylase induction was diminished. In contrast, similar treatments with Puromycin or Cycloheximide did not enhance ornithine decarboxylase above serum or dibutyryl CAMP stimulated levels, but always attenuated its induction at all time intervals tested. Dibutyryl CAMP induced ornithine decarboxylase above the serum stimulated levels only when added during the initial 2 hrs. after stimulation with serum. These data and other observations presented suggest that transcriptional processes leading to the induction of ornithine decarboxylase occur primarily during the initial 2 hrs. after refeeding with serum and that the major action of dibutyryl CAMP in the induction of this enzyme appears to be at pretranslational sites. Ornithine the
rate
limiting
in almost enzyme tion
decarboxylase
all
enzyme for tissues
(2,3).
This
of tissues
protein
(ODC) (L-ornithine biosynthesis
and has the shortest enzyme
presumably
synthesis
the
is
rapidly
the CAMP stimulated
activity
is
enhanced Clark
(9)
reported
potentiated
the
on the
administration activity
of antibody
3T3 cells. stimulated
due to dibutyryl
0006-291X/78/0813-0832$01,00/0 Copyright 0 I978 by Academic Press, Inc. AN rights of reproduction in any form reserved.
832
is
found
growth
and prolifera-
a role
in RNA and
translational
in a variety
control of tissues
to inhibitors
precipitable
of since
of both
ODC molecules
(6-8).
superinduction
to rats
1.14)
It is
(1).
play
is sensitive
CAMP elevation
in serum stimulated
Puromycin
and/or
enzyme
and formation
by intracellular
by Cordycepin that
synthesis
of this
the
which
mechanism
4.1
of any known mamnalian
during
The transcriptional
by a CAMP-dependent
RNA and protein
half-life
polyamines
ODC may be mediated
E.C.
of polyamines
induced
to produce
(4,5).
carboxylase
(dB)
of ODC by Actinomycin
D but not
Beck et -- al.
reported
hepatic
(10)
have
ODC activity
CAMP administration.
and In the
BIOCHEMICAL AND BIOWYSICAL RESEARCH COMMUNICATIONS
Vol. 81, No: 3, 1978
present
study,
duction
of ODC by serum
hamster
ovary
times
the effects
These
inhibitors
leading
to the
investigated
on the using
to understand
induction
MATERIALS
inhibitors
and dB CAMP were
in an attempt
serum refeeding,
on processes
synthesis
or serum and dB CAMP were
(CHO) cells.
following
effects
of RNA and protein
Chinese
added their
in-
at various temporal
of ODC.
AND METHODS
CHO cells were maintained in monolayer cultures as previously described (11-13) and routinely checked for mycoplasma contamination by the method of Peden (14). Confluent cultures were placed in serum free McCoys 5a media for 16-18 hrs. The cells were stimulated with fresh McCoys 5a media (15) containing 20% fetal bovine serum (Gibco, Inc.) in the absence and presence of dB CAMP. At various times after this stimulation cells were scraped with a rubber policeman from plates, (15Ox20mm) collected by centrifugation, and homogenized by sonication in 0.70 ml of 0.05 M phosphate buffer pH 7.2 containing 1mM dithiothreitol and O.lmM EDTA. The homogenate was centrifuged at 50,000 xg for 10 min. and 0.17 ml of the resulting supernatant solution was used for assay. Ornithine decarboxylase activity was assessed by measuring the liberation of 14c02 from [l-14C] DL-ornithine (New England Nuclear 40-50 m Ci/m mol). The reaction contained 085 M phosphate buffer pH 7.2, 25~M pyridoxyl phosphate, 1mM dithiothreitol, lmM[l-14C] L-ornithine (containing 1.25 cpm/pmol L-ornithine) O.lmM EDTA in a final volume of 0.20 ml. The reaction was carried out in 15 ml conical glass centrifuge tubes, which were sealed with a rubber stopper supporting a polyethylene well containing a strip of Whatman 3MM filter paper, impregnated with 50uL of Protosol (New England Nuclear, 0.5 M). The reaction was allowed to proceed at 37'C for 60 min. and was terminated by injection of 2 ml of 1 M citric acid through the wells. The tubes were incubated at 37°C for an additional 30 min. and then the filter paper was counted in 5 ml of an Omnifluortoluene mixture using a Packard liquid scintillation counter. In some experiments, cells were pulsed for 30 min. with [3H] thymidine or [3H] leucine or [3H] uridine around the time intervals shown in the tables. The incorporation of these radiolabeled precursors into trichloroacetic acid insoluble material was determined by Millipore filtration. RESULTS Increases
in ODC activity
were
serum or serum and dB CAMP (Table stimulation
and enzyme activity
refeeding.
The simultaneous
2 fold
above the
refeeding increase
with
in thymidine
by serum or serum Cycloheximide, media
at 0 hrs.
1).
addition
a period uptake
containing
or Puromycin (Table
2).
2 hrs.
Maximal
declined
serum stimulated
serum,
apparent
to basal
stimu lation were
found
levels
5-6
of dB CAMP and serum enhanced (Table
of DNA synthesis 1).
or Actinomycin if
833
1)
From 5-7
occurred
The increase
dB CAMP is markedly
However,
ODC levels
rapidly
levels.
(Table
following
diminished
D or Cordycepin these
agents
4 hrs. hrs.
after after
ODC activity
hrs.following
as suggested activity
with
by the
of ODC produced by placing
in the
are placed
either
stimulation
in the media
at
Vol. 81,No.3,
1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 1 CHANGES
IN ORNITHINE
DECARBOXVLASE
FOLLOWING Time after stimulation (hrs.)
ACTIVITY
AND THVMIDINE
UPTAKE
SERUM AND dB CAMP STIMULATION Serum
Serum
ODC Activity Co2/60 min /mg protein)
(nmol
[$H methylj thpidine uptake (cpm/lO cells)
and dR c&VP OOC Activity Co 2/60 min /mg protein)
(nmol I
0
0.231
f 0.031
1,216
0.289
f 0.011
1
0.242
k 0.014
1,304
0.311
?. 0.021
2
0.821
i 0.052
1,373
1.013
+ 0.061
3
1.013
t 0.073
1,510
1.928
f 0.087
4
1.261
t 0.113
1,492
2.413
f 0.058
5
0.538
f 0.032
4,312
0.512
f 0.061
6
0.401
i 0.017
7,039
0.358
f 0.022
7
0.231
+ 0.015
10,321
0.316
+ 0.016
a
0.301
_+ 0.022
11,191
0.333
_+ 0.029
9
0.341
t 0.016
10,873
0.288
t 0.011
Confluent aDnolayers of CHO cells were placed in serum free McCoy's 5a media (15) for 16-18 hrs. The cultures were then fed with fresh media containing 20% fetal bovine serum in the absence and presence of 1mM dB CAMP. Enzyme activity was assessed at hourly intervals. (See Methods section for details.) Cultures were also pulsed with lrci/ml of C3H-methyl] thymidine for 30 min around the time intervals shown in the table and the incorporation of radiolabel percursor into trichloroacetic acid insoluble material was determined by millipore filtration. Each nuder for ODC activity represents the mean ?r S.E.Y. of 3 determinations for each of 2 separate plates. The [3H-methyl]thymidine incorporation is the mean of 3 determinations frum 1 plate.
various
times
example,
after
by 1.5-2
or Cordycepin this the
the hrs.
2 hrs.
stimulation,
after
no longer
initial
initial
stimulation Cordycepin
when Puromycin (Table was found
the enzyme assayed The superinduction tration
dependent,
dB CAMP (Table
3).
2).
ODC activity
addition
serum or dB CAMP stimulated
depressed
of these levels
Optimal when these
(Table were
superinduction drugs
(Table
were
the
Enhancement
Cordycepin
was also
concentration
depressed
by addition
of these
added
at all
In fact,
points
of ODC by Actinomycin added
3 hrs.
above
ODC activity time
after
D
after
ODC activity
In contrast,
2).
Actinomycin
at 4 hrs. enhanced
For
was
after
D or
stimulation
and
2).
of ODC by either whether
assayed
two agents
or Cycloheximide
at 4 hrs
seen differed.
serum or serum and dB CAMP stimulation,
depressed
the
the effects
cells
Actinomycin
were
of ['H]
stimulated leucine
dependent, drugs
at 3 hrs.
834
D or Cordycepin
by serum or serum containing
uptake while
was concen-
by Actinomycin
C3H] uridine
following
serum
D and
uptake stimulation
was markedly (Table
3).
BlOCi-lEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 8 1, No, 3,1978
TABLE 2 EFFECT OF INHIBITORS AND dBcAMP
OF RNA AND PROTEIN
STIMJLATION
OF ORNITHINE
Time Inhibitor
Interval
SYNTHESIS
ON THE SERUM
DECARBOXYLASE
ACTIVITY
Drnithine Decarboxylase (nmol Co2 liberated/60
(hrs)
Serum
Activity mln/mg protein) Serum
I
and
dBcAMP
Cycloheximide
Puromycin (50 ugfml)
Actinomycfn (4 ugfml)
O-4.0 . - . . - . . - .
0.066 0.091 0.267 0.711
0.060 0.113 0.419 1.238
O-4.0 . - . . - . . - . 9 C-d n &.a--.”
0.238 0.999 1.781 1.952 1I.--.ar. oc9 2.014 2.142 1.914
0.317 2.332 2.965 3.356 7.J..s.s” ?*k 4.123 5.319 3.270
D
,
-
.
. - . . - .
-
Cordycepin (25 ug/ml)
Control
(1)
No inhibitors (O-4 hrs)
1.411
2.631
Control
(2)
No inhibitors (O-4 hrs)
1.321
2.512
Confluent cultures were stimulated with serum In the absence and presence of dBcAHP (111#4, see Methods). The cultures were incubated with the drugs shown in the table for the time intervals after serum stimulation indicated in the table. ODC activity was assessed 4 hrs. after serum stimulation as described in the legend to Each nuker shown in the table represents the mean of 3 determinations Table 1. S.E.M.,for the ntiers shown in the table (when expressed from 2 separate plates. as a percentage of the man) ranged from 3.21% - 11.68%.
Cycloheximide about
inhibited
the levels the
leucine
uptake
about
90% and C3H] uridine
uptake
by
40%. The enhancement
agent
[3H]
was added initial
initial
The stimulation
(Table
2 hrs.
produced
of ODC activity 4).
after
by serum
2 hrs.
by dB CAMP depended
For example, serum stimulation, alone
no enhancement
of ODC activity
(Table
4).
if
dB CAMP was placed ODC activity However,
of ODC activity by dB CAMP occurred
835
on the time
if
was found only
this
in the media within
was elevated this
at which
agent
above the
was added
at 4 hrs.
when added
(Table during
after 4). the initia
Vol. 8 l,No.3,1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 3 EFFECTS OF CORDYCEPIN DECARBOXYLASE
ACTIVITY,
AND ACTINOMYCIN [3H]
LEUCINE
D ON ORNITHINE
AND [3H]
URIDINE
UPTAKE
Drnithine C3Hip::;:ine Drug
cpm/106
No drug
452
ActinoaBcin (0.5 m/ml
Uridine Uptake cpm/lo6 cells
Activity
cells i 32
2934
nmol
(Q/60
min
Serum
i 121
1.321
/mg'p rotein
Serum
and
dBcAMP
t 0.071
2.432
i 0.168
D 1
514 f 41
---
1.532
f 0.083
3.096
f 0.092
1.0
g/ml
623
---
1.678
* 0.042
3.824
f 0.171
2.0
g/ml
678 f
* 0.041
4.321
+ 0.162
4.0
g/ml
8.0
g/ml
f 22 30
212
f
16
1.937
709
f 43
203
f 12
2.148
i: 0.053
5.413
* 0.189
804
f 16
---
2.271
f 0.062
6.164
f 0.203
Cordycepin 1 uglml
672
* 27
_--
2.212
+ 0.092
3.892
+ 0.177
5 Pg/ml
778
+ 43
862
*47
2.810
f 0.103
4.913
f 0.236
829 f 38
412
f 22
3.103
+ 0.079
5.532
+ 0.168
1821
f 111
0.712
+ 0.102
1.103
* 0.049
25 Pg/ml Cycloheximide (25 ug/ml)
46+
5
Confluent serum deprived cultures were stimulated measurements the cultures were stimulated with serum dB CAMP. At 3 hrs. after this stimulation the drugs and ODC activity determined 1 hr. later (see Methods In s me cases the cells were pulsed for 3.5-4-O hrs. s or [ I+] uridlne. The incorporation of these isotopes insoluble material was determined by Millipore filtration. the man of 3 determinations from 3 separate plates. that these conditions were not tested.
2 hrs., the
Dac;~~-vvse
r3H]
even
initial
if
enzyme activity
stimulation
with
with fresh serum. in the absence and shown in the table section for assay with O.ZPCi/ml of into trichloroacetic Each number The dashed lines
was determined
For ODC presence of were added rocedures). f 3H] Leucine acid represents indicate
at 5 or 6 or 7 hrs.
following
serum.
DISCUSSION A number
of interesting
of CAMP-mediated ted. are
induction
The concept primed
for
4.
Only
Table
induction events
concepts
of ODC in synchronous
of a critical
the during
time
CAMP-mediated this
of ODC above the are occurring
and considerations
during
effects
of RNA and protein
effects
of Actinomycin
interval
induction
critical
time
time
synthesis
(early is
levels.
interval inhibitors
D and Cordycepin
836
Gl) where
did
addition
is
the mechanisms
proliferation
suggested
interval
serum stimulated this
cell
about
by the
are presencellular data
dB CAMP enhance The concept
further
suggested
on ODC activity. prior
that
events shown
in
the critical by the
The opposite
to and following
this
BIOCHEMICAL
Vol. 81, No. 3, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE
4
Enhancement of Omithine Decarboxy'lase dB CAMP Addition at Various Time Following Serum Stimulation
Serum
4.0
3.00
0.5
u
4.0
2.88
1.0
+-+ 4.0
3.11
1.5
ct
4.0
2.89
2.0
ct
4.0
2.99
2.5
++ 4.0
1.91
3.0
*
4.0
1.21
3.5
++ 4.0
1.31
(O-5.0)
0.61
0 *
5.0
1.18
1.0
*
5.0
1.22
1.5
++ 5.0
1.30
2.0
4-P 5.0
1.07
3.0
*
5.0
0.53
4.0
*
5.0
0.36
Serum
(O-6.0) 0 -
0.34
6.0
0.47
1.0
++ 6.0
0.56
1.5
u
6.0
0.61
2.0
-
6.0
0.53
2.5
++ 6.0
0.27
3.0
-
6.0
0.23
4.0
u
6.0
0.14
5.0
-
6.0
0.28
Serum
(O-7.0) D N
0.28
7.0
0.32
3.0
++. 7.0
0.24
4.0
cc
0.26
7.0
Confluent These
cultures
fetal
bovine
comittant
time
transcriptional
were
various
represents
interval levels
with
Some of the
serum
addftion prior
at 4, the
(see Table during
other to
early
the
serum McCoys
cultures
5, 6 and
mean of
were fresh
while
times
was assessed point
of CHO cells
stimulated
serum.
for
activity Each
cultures
with
incubate
Activity Protein
1.39
(D-4.0) 0 -
Serum
critical
by
Ornithine Decarboxylase nmol CO, Liberated/60min/mg
Interval (hrs.) dB CAMP (TM]
Time with
Activity Intervals
for
5a media
containfng
11W4 dB CAMP conwere
addition after from
allowed
of dB CAMP. serum 1 culture
that
G, while
after
hrs. 20%
received
2) suggest
837
16-18
cultures
7 hrs.
3 assays
deprived
to Enzyme
stimulation. dish
of
ODC activity this
time
cells.
is regulated interval
the
at events
Vol. 8 1, No. 3, 1978
BIOCHEMICAL
regulating
ODC activity
are
Cordycepin
may involve
similar
as depression perhaps since,
either
intervals
Puromycin
--in vitro
Cordycepin
cell
criptional induce only
level ODC.
act
An examination
during
this
cell
cycle
during
this
same time
kinase
is
a small
during
amount
this
type
II
action
time
kinase,
it
is
which
in turn While
kinase
enhances
these
events
significantly
to the
involvement
the
kinase
thought
protein
is
during protein
at the
to why CAMP can may relate kinase protein
activity
form.
G1, the
Since
the major
of CAMP-dependent
interval
CAMP activates
which
then
may phosphorylate
nuclear
is
expressed
from mid to late
activation
trans-
G, and there
kinase
time
protein
type
I
proteins
(18,19),
of ODC.
of ODC, other
important
yet
unknown
of ODC as well. induction
protein
D or
protein
early
this
of CAMP in the
uptake
investigators
ODC induction,
during
induction
Other
determine
the predominant
transcription
leucine
I CAMP-dependent
period,
to be the
seem to be very
I CAMP-dependent
present
time
[3H]
might
or
time
serum stimulation
The type
this
at all
I CAMP-dependent
CAMP-dependent
protein is
of type
(9)
interesting
CAMP acts
to enhance
kinase
After
that
induction
of type
II
interval.
CAMP-mediated
the
form of
suggested
CAMP-dependent
events
(ll-13,16).
of type
of CAMP in cells
that
related
interval
CAMP-dependent
suggest
of what
expression
the predominant
(17).
following
such
by Actinomycin
systems
interval
is
3).
synthesis
2 hrs.
time
specific
enhanced
initial
critical
to the
only
the
point
(Table
of protein
D and
of the enzyme
ODC activity
serum stimulation
above observations
during
half-life
depressed
synthesis
of Actinomycin
to "superinduction"
D or Cordycepin
protein
the
effects
The latter
or Cycloheximide
free
Collectively,
increased
stimulation
RESEARCH COMMUNICATIONS
attributed
synthesis.
following
have observed in
(lo),
and Actinomycin
at 3 hrs.
and the
processes
of protein
tested
when added
more complex
of an inhibitor
stimulation
AND BIOPHYSICAL
kinase
and closely processes However,
of ODC should regulating
linked
to the
may contribute continued
focus
studies
of
on the possible
the transcription
role
of ODC.
ACKNOWLEDGEMENTS The excellent
technical
assistance
838
of Ms. Julie
Nye is
gratefully
acknowledged.
Vol. 81, No. 3, 1978
8lOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
This
by Research
work
was supported
Institute from
of Environmental
the Energy
provided
Health
Research
by a grant
1.
Tabor,
H. and Tabor,
2.
Manen,
C. A. and Russell,
3.
Russell,
5.
Sciences
of Materials
from
and by Research
Research
the National
Grant
Administration.
from the Connecticut
Institute
4.
#E.S.-0133702
and Development
the
#E(ll-l)-3140
Additional
Foundation
support
and by funds
was from
Science.
C. W.
(1972) D. H.
D. H. and Snyder,
U.S.A.,
Grant
Adv.
in Enz.
(1975)
S. H.
Life
(1968)
3&, 203-268.
Sci.
Proc.
lJ,
1769-1776.
Nat.
Acad.
Sci.,
60-, 1420-1427.
Bachrach,
U.
(1973)
Press,
New York.
Cohen,
S. S.
Function
(1971)
of Naturally
Introduction
Occurring
Polyamine,
to the Polyamines,
Academic
Prentice-Hall,
New Jersey. 6.
Canellakis,
Z. N. and Theoharides,
T. C.
(1976)
J. Biol.
Chem.,
251,
4436-4441. 7.
Beck,
W. T.,
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 832-840
SUPERINDUCTION OF ORNITHINE DECARBOXYLASE BY ACTINOMYCIN D AND CORDYCEPIN Max Costa Institute of Materials Science and Department of Laboratory Medicine University of Connecticu't Storrs, Connecticut 06268
Received
December 19,1977
Cordycepin and Actinomycin D when added 2.0 - 3.5 hrs. following the addition of serum or serum and dibutyryl CAMP to quiescent cultures of Chinese hamster ovary cells, resulted in "superinduction" of ornithine decarboxylase at 4 hrs. If these agents were added from 0 to 2 hrs after stimulation, ornithine decarboxylase induction was diminished. In contrast, similar treatments with Puromycin or Cycloheximide did not enhance ornithine decarboxylase above serum or dibutyryl CAMP stimulated levels, but always attenuated its induction at all time intervals tested. Dibutyryl CAMP induced ornithine decarboxylase above the serum stimulated levels only when added during the initial 2 hrs. after stimulation with serum. These data and other observations presented suggest that transcriptional processes leading to the induction of ornithine decarboxylase occur primarily during the initial 2 hrs. after refeeding with serum and that the major action of dibutyryl CAMP in the induction of this enzyme appears to be at pretranslational sites. Ornithine the
rate
limiting
in almost enzyme tion
decarboxylase
all
enzyme for tissues
(2,3).
This
of tissues
protein
(ODC) (L-ornithine biosynthesis
and has the shortest enzyme
presumably
synthesis
the
is
rapidly
the CAMP stimulated
activity
is
enhanced Clark
(9)
reported
potentiated
the
on the
administration activity
of antibody
3T3 cells. stimulated
due to dibutyryl
0006-291X/78/0813-0832$01,00/0 Copyright 0 I978 by Academic Press, Inc. AN rights of reproduction in any form reserved.
832
is
found
growth
and prolifera-
a role
in RNA and
translational
in a variety
control of tissues
to inhibitors
precipitable
of since
of both
ODC molecules
(6-8).
superinduction
to rats
1.14)
It is
(1).
play
is sensitive
CAMP elevation
in serum stimulated
Puromycin
and/or
enzyme
and formation
by intracellular
by Cordycepin that
synthesis
of this
the
which
mechanism
4.1
of any known mamnalian
during
The transcriptional
by a CAMP-dependent
RNA and protein
half-life
polyamines
ODC may be mediated
E.C.
of polyamines
induced
to produce
(4,5).
carboxylase
(dB)
of ODC by Actinomycin
D but not
Beck et -- al.
reported
hepatic
(10)
have
ODC activity
CAMP administration.
and In the
BIOCHEMICAL AND BIOWYSICAL RESEARCH COMMUNICATIONS
Vol. 81, No: 3, 1978
present
study,
duction
of ODC by serum
hamster
ovary
times
the effects
These
inhibitors
leading
to the
investigated
on the using
to understand
induction
MATERIALS
inhibitors
and dB CAMP were
in an attempt
serum refeeding,
on processes
synthesis
or serum and dB CAMP were
(CHO) cells.
following
effects
of RNA and protein
Chinese
added their
in-
at various temporal
of ODC.
AND METHODS
CHO cells were maintained in monolayer cultures as previously described (11-13) and routinely checked for mycoplasma contamination by the method of Peden (14). Confluent cultures were placed in serum free McCoys 5a media for 16-18 hrs. The cells were stimulated with fresh McCoys 5a media (15) containing 20% fetal bovine serum (Gibco, Inc.) in the absence and presence of dB CAMP. At various times after this stimulation cells were scraped with a rubber policeman from plates, (15Ox20mm) collected by centrifugation, and homogenized by sonication in 0.70 ml of 0.05 M phosphate buffer pH 7.2 containing 1mM dithiothreitol and O.lmM EDTA. The homogenate was centrifuged at 50,000 xg for 10 min. and 0.17 ml of the resulting supernatant solution was used for assay. Ornithine decarboxylase activity was assessed by measuring the liberation of 14c02 from [l-14C] DL-ornithine (New England Nuclear 40-50 m Ci/m mol). The reaction contained 085 M phosphate buffer pH 7.2, 25~M pyridoxyl phosphate, 1mM dithiothreitol, lmM[l-14C] L-ornithine (containing 1.25 cpm/pmol L-ornithine) O.lmM EDTA in a final volume of 0.20 ml. The reaction was carried out in 15 ml conical glass centrifuge tubes, which were sealed with a rubber stopper supporting a polyethylene well containing a strip of Whatman 3MM filter paper, impregnated with 50uL of Protosol (New England Nuclear, 0.5 M). The reaction was allowed to proceed at 37'C for 60 min. and was terminated by injection of 2 ml of 1 M citric acid through the wells. The tubes were incubated at 37°C for an additional 30 min. and then the filter paper was counted in 5 ml of an Omnifluortoluene mixture using a Packard liquid scintillation counter. In some experiments, cells were pulsed for 30 min. with [3H] thymidine or [3H] leucine or [3H] uridine around the time intervals shown in the tables. The incorporation of these radiolabeled precursors into trichloroacetic acid insoluble material was determined by Millipore filtration. RESULTS Increases
in ODC activity
were
serum or serum and dB CAMP (Table stimulation
and enzyme activity
refeeding.
The simultaneous
2 fold
above the
refeeding increase
with
in thymidine
by serum or serum Cycloheximide, media
at 0 hrs.
1).
addition
a period uptake
containing
or Puromycin (Table
2).
2 hrs.
Maximal
declined
serum stimulated
serum,
apparent
to basal
stimu lation were
found
levels
5-6
of dB CAMP and serum enhanced (Table
of DNA synthesis 1).
or Actinomycin if
833
1)
From 5-7
occurred
The increase
dB CAMP is markedly
However,
ODC levels
rapidly
levels.
(Table
following
diminished
D or Cordycepin these
agents
4 hrs. hrs.
after after
ODC activity
hrs.following
as suggested activity
with
by the
of ODC produced by placing
in the
are placed
either
stimulation
in the media
at
Vol. 81,No.3,
1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 1 CHANGES
IN ORNITHINE
DECARBOXVLASE
FOLLOWING Time after stimulation (hrs.)
ACTIVITY
AND THVMIDINE
UPTAKE
SERUM AND dB CAMP STIMULATION Serum
Serum
ODC Activity Co2/60 min /mg protein)
(nmol
[$H methylj thpidine uptake (cpm/lO cells)
and dR c&VP OOC Activity Co 2/60 min /mg protein)
(nmol I
0
0.231
f 0.031
1,216
0.289
f 0.011
1
0.242
k 0.014
1,304
0.311
?. 0.021
2
0.821
i 0.052
1,373
1.013
+ 0.061
3
1.013
t 0.073
1,510
1.928
f 0.087
4
1.261
t 0.113
1,492
2.413
f 0.058
5
0.538
f 0.032
4,312
0.512
f 0.061
6
0.401
i 0.017
7,039
0.358
f 0.022
7
0.231
+ 0.015
10,321
0.316
+ 0.016
a
0.301
_+ 0.022
11,191
0.333
_+ 0.029
9
0.341
t 0.016
10,873
0.288
t 0.011
Confluent aDnolayers of CHO cells were placed in serum free McCoy's 5a media (15) for 16-18 hrs. The cultures were then fed with fresh media containing 20% fetal bovine serum in the absence and presence of 1mM dB CAMP. Enzyme activity was assessed at hourly intervals. (See Methods section for details.) Cultures were also pulsed with lrci/ml of C3H-methyl] thymidine for 30 min around the time intervals shown in the table and the incorporation of radiolabel percursor into trichloroacetic acid insoluble material was determined by millipore filtration. Each nuder for ODC activity represents the mean ?r S.E.Y. of 3 determinations for each of 2 separate plates. The [3H-methyl]thymidine incorporation is the mean of 3 determinations frum 1 plate.
various
times
example,
after
by 1.5-2
or Cordycepin this the
the hrs.
2 hrs.
stimulation,
after
no longer
initial
initial
stimulation Cordycepin
when Puromycin (Table was found
the enzyme assayed The superinduction tration
dependent,
dB CAMP (Table
3).
2).
ODC activity
addition
serum or dB CAMP stimulated
depressed
of these levels
Optimal when these
(Table were
superinduction drugs
(Table
were
the
Enhancement
Cordycepin
was also
concentration
depressed
by addition
of these
added
at all
In fact,
points
of ODC by Actinomycin added
3 hrs.
above
ODC activity time
after
D
after
ODC activity
In contrast,
2).
Actinomycin
at 4 hrs. enhanced
For
was
after
D or
stimulation
and
2).
of ODC by either whether
assayed
two agents
or Cycloheximide
at 4 hrs
seen differed.
serum or serum and dB CAMP stimulation,
depressed
the
the effects
cells
Actinomycin
were
of ['H]
stimulated leucine
dependent, drugs
at 3 hrs.
834
D or Cordycepin
by serum or serum containing
uptake while
was concen-
by Actinomycin
C3H] uridine
following
serum
D and
uptake stimulation
was markedly (Table
3).
BlOCi-lEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 8 1, No, 3,1978
TABLE 2 EFFECT OF INHIBITORS AND dBcAMP
OF RNA AND PROTEIN
STIMJLATION
OF ORNITHINE
Time Inhibitor
Interval
SYNTHESIS
ON THE SERUM
DECARBOXYLASE
ACTIVITY
Drnithine Decarboxylase (nmol Co2 liberated/60
(hrs)
Serum
Activity mln/mg protein) Serum
I
and
dBcAMP
Cycloheximide
Puromycin (50 ugfml)
Actinomycfn (4 ugfml)
O-4.0 . - . . - . . - .
0.066 0.091 0.267 0.711
0.060 0.113 0.419 1.238
O-4.0 . - . . - . . - . 9 C-d n &.a--.”
0.238 0.999 1.781 1.952 1I.--.ar. oc9 2.014 2.142 1.914
0.317 2.332 2.965 3.356 7.J..s.s” ?*k 4.123 5.319 3.270
D
,
-
.
. - . . - .
-
Cordycepin (25 ug/ml)
Control
(1)
No inhibitors (O-4 hrs)
1.411
2.631
Control
(2)
No inhibitors (O-4 hrs)
1.321
2.512
Confluent cultures were stimulated with serum In the absence and presence of dBcAHP (111#4, see Methods). The cultures were incubated with the drugs shown in the table for the time intervals after serum stimulation indicated in the table. ODC activity was assessed 4 hrs. after serum stimulation as described in the legend to Each nuker shown in the table represents the mean of 3 determinations Table 1. S.E.M.,for the ntiers shown in the table (when expressed from 2 separate plates. as a percentage of the man) ranged from 3.21% - 11.68%.
Cycloheximide about
inhibited
the levels the
leucine
uptake
about
90% and C3H] uridine
uptake
by
40%. The enhancement
agent
[3H]
was added initial
initial
The stimulation
(Table
2 hrs.
produced
of ODC activity 4).
after
by serum
2 hrs.
by dB CAMP depended
For example, serum stimulation, alone
no enhancement
of ODC activity
(Table
4).
if
dB CAMP was placed ODC activity However,
of ODC activity by dB CAMP occurred
835
on the time
if
was found only
this
in the media within
was elevated this
at which
agent
above the
was added
at 4 hrs.
when added
(Table during
after 4). the initia
Vol. 8 l,No.3,1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 3 EFFECTS OF CORDYCEPIN DECARBOXYLASE
ACTIVITY,
AND ACTINOMYCIN [3H]
LEUCINE
D ON ORNITHINE
AND [3H]
URIDINE
UPTAKE
Drnithine C3Hip::;:ine Drug
cpm/106
No drug
452
ActinoaBcin (0.5 m/ml
Uridine Uptake cpm/lo6 cells
Activity
cells i 32
2934
nmol
(Q/60
min
Serum
i 121
1.321
/mg'p rotein
Serum
and
dBcAMP
t 0.071
2.432
i 0.168
D 1
514 f 41
---
1.532
f 0.083
3.096
f 0.092
1.0
g/ml
623
---
1.678
* 0.042
3.824
f 0.171
2.0
g/ml
678 f
* 0.041
4.321
+ 0.162
4.0
g/ml
8.0
g/ml
f 22 30
212
f
16
1.937
709
f 43
203
f 12
2.148
i: 0.053
5.413
* 0.189
804
f 16
---
2.271
f 0.062
6.164
f 0.203
Cordycepin 1 uglml
672
* 27
_--
2.212
+ 0.092
3.892
+ 0.177
5 Pg/ml
778
+ 43
862
*47
2.810
f 0.103
4.913
f 0.236
829 f 38
412
f 22
3.103
+ 0.079
5.532
+ 0.168
1821
f 111
0.712
+ 0.102
1.103
* 0.049
25 Pg/ml Cycloheximide (25 ug/ml)
46+
5
Confluent serum deprived cultures were stimulated measurements the cultures were stimulated with serum dB CAMP. At 3 hrs. after this stimulation the drugs and ODC activity determined 1 hr. later (see Methods In s me cases the cells were pulsed for 3.5-4-O hrs. s or [ I+] uridlne. The incorporation of these isotopes insoluble material was determined by Millipore filtration. the man of 3 determinations from 3 separate plates. that these conditions were not tested.
2 hrs., the
Dac;~~-vvse
r3H]
even
initial
if
enzyme activity
stimulation
with
with fresh serum. in the absence and shown in the table section for assay with O.ZPCi/ml of into trichloroacetic Each number The dashed lines
was determined
For ODC presence of were added rocedures). f 3H] Leucine acid represents indicate
at 5 or 6 or 7 hrs.
following
serum.
DISCUSSION A number
of interesting
of CAMP-mediated ted. are
induction
The concept primed
for
4.
Only
Table
induction events
concepts
of ODC in synchronous
of a critical
the during
time
CAMP-mediated this
of ODC above the are occurring
and considerations
during
effects
of RNA and protein
effects
of Actinomycin
interval
induction
critical
time
time
synthesis
(early is
levels.
interval inhibitors
D and Cordycepin
836
Gl) where
did
addition
is
the mechanisms
proliferation
suggested
interval
serum stimulated this
cell
about
by the
are presencellular data
dB CAMP enhance The concept
further
suggested
on ODC activity. prior
that
events shown
in
the critical by the
The opposite
to and following
this
BIOCHEMICAL
Vol. 81, No. 3, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE
4
Enhancement of Omithine Decarboxy'lase dB CAMP Addition at Various Time Following Serum Stimulation
Serum
4.0
3.00
0.5
u
4.0
2.88
1.0
+-+ 4.0
3.11
1.5
ct
4.0
2.89
2.0
ct
4.0
2.99
2.5
++ 4.0
1.91
3.0
*
4.0
1.21
3.5
++ 4.0
1.31
(O-5.0)
0.61
0 *
5.0
1.18
1.0
*
5.0
1.22
1.5
++ 5.0
1.30
2.0
4-P 5.0
1.07
3.0
*
5.0
0.53
4.0
*
5.0
0.36
Serum
(O-6.0) 0 -
0.34
6.0
0.47
1.0
++ 6.0
0.56
1.5
u
6.0
0.61
2.0
-
6.0
0.53
2.5
++ 6.0
0.27
3.0
-
6.0
0.23
4.0
u
6.0
0.14
5.0
-
6.0
0.28
Serum
(O-7.0) D N
0.28
7.0
0.32
3.0
++. 7.0
0.24
4.0
cc
0.26
7.0
Confluent These
cultures
fetal
bovine
comittant
time
transcriptional
were
various
represents
interval levels
with
Some of the
serum
addftion prior
at 4, the
(see Table during
other to
early
the
serum McCoys
cultures
5, 6 and
mean of
were fresh
while
times
was assessed point
of CHO cells
stimulated
serum.
for
activity Each
cultures
with
incubate
Activity Protein
1.39
(D-4.0) 0 -
Serum
critical
by
Ornithine Decarboxylase nmol CO, Liberated/60min/mg
Interval (hrs.) dB CAMP (TM]
Time with
Activity Intervals
for
5a media
containfng
11W4 dB CAMP conwere
addition after from
allowed
of dB CAMP. serum 1 culture
that
G, while
after
hrs. 20%
received
2) suggest
837
16-18
cultures
7 hrs.
3 assays
deprived
to Enzyme
stimulation. dish
of
ODC activity this
time
cells.
is regulated interval
the
at events
Vol. 8 1, No. 3, 1978
BIOCHEMICAL
regulating
ODC activity
are
Cordycepin
may involve
similar
as depression perhaps since,
either
intervals
Puromycin
--in vitro
Cordycepin
cell
criptional induce only
level ODC.
act
An examination
during
this
cell
cycle
during
this
same time
kinase
is
a small
during
amount
this
type
II
action
time
kinase,
it
is
which
in turn While
kinase
enhances
these
events
significantly
to the
involvement
the
kinase
thought
protein
is
during protein
at the
to why CAMP can may relate kinase protein
activity
form.
G1, the
Since
the major
of CAMP-dependent
interval
CAMP activates
which
then
may phosphorylate
nuclear
is
expressed
from mid to late
activation
trans-
G, and there
kinase
time
protein
type
I
proteins
(18,19),
of ODC.
of ODC, other
important
yet
unknown
of ODC as well. induction
protein
D or
protein
early
this
of CAMP in the
uptake
investigators
ODC induction,
during
induction
Other
determine
the predominant
transcription
leucine
I CAMP-dependent
period,
to be the
seem to be very
I CAMP-dependent
present
time
[3H]
might
or
time
serum stimulation
The type
this
at all
I CAMP-dependent
CAMP-dependent
protein is
of type
(9)
interesting
CAMP acts
to enhance
kinase
After
that
induction
of type
II
interval.
CAMP-mediated
the
form of
suggested
CAMP-dependent
events
(ll-13,16).
of type
of CAMP in cells
that
related
interval
CAMP-dependent
suggest
of what
expression
the predominant
(17).
following
such
by Actinomycin
systems
interval
is
3).
synthesis
2 hrs.
time
specific
enhanced
initial
critical
to the
only
the
point
(Table
of protein
D and
of the enzyme
ODC activity
serum stimulation
above observations
during
half-life
depressed
synthesis
of Actinomycin
to "superinduction"
D or Cordycepin
protein
the
effects
The latter
or Cycloheximide
free
Collectively,
increased
stimulation
RESEARCH COMMUNICATIONS
attributed
synthesis.
following
have observed in
(lo),
and Actinomycin
at 3 hrs.
and the
processes
of protein
tested
when added
more complex
of an inhibitor
stimulation
AND BIOPHYSICAL
kinase
and closely processes However,
of ODC should regulating
linked
to the
may contribute continued
focus
studies
of
on the possible
the transcription
role
of ODC.
ACKNOWLEDGEMENTS The excellent
technical
assistance
838
of Ms. Julie
Nye is
gratefully
acknowledged.
Vol. 81, No. 3, 1978
8lOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
This
by Research
work
was supported
Institute from
of Environmental
the Energy
provided
Health
Research
by a grant
1.
Tabor,
H. and Tabor,
2.
Manen,
C. A. and Russell,
3.
Russell,
5.
Sciences
of Materials
from
and by Research
Research
the National
Grant
Administration.
from the Connecticut
Institute
4.
#E.S.-0133702
and Development
the
#E(ll-l)-3140
Additional
Foundation
support
and by funds
was from
Science.
C. W.
(1972) D. H.
D. H. and Snyder,
U.S.A.,
Grant
Adv.
in Enz.
(1975)
S. H.
Life
(1968)
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Sci.
Proc.
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1769-1776.
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U.
(1973)
Press,
New York.
Cohen,
S. S.
Function
(1971)
of Naturally
Introduction
Occurring
Polyamine,
to the Polyamines,
Academic
Prentice-Hall,
New Jersey. 6.
Canellakis,
Z. N. and Theoharides,
T. C.
(1976)
J. Biol.
Chem.,
251,
4436-4441. 7.
Beck,
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