Vol. 81, No. 3, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
April 14,1978
Pages 1054-1057
ORNITHINE DECARBOXYLASE INDUCTION AND NUCLEOLAR RNA SYNTHESIS IN FRIEND LEUKEMIA CELLS P. J. Department
Dehlinger
of Biochemistry
and Division
School University
Received
March
and M. Litt of Medical
Genetics
of Medicine
of Oregon
Health
Sciences
Portland,
Oregon
97201
Center
1, 1978
The induction
of ornithine decarboxylase and the stimulation of following dilution of stationary phase Friend Leukemia Cells into fresh medium were studied. Ornithine decarboxylase activity and the rate of nucleolar RNA synthesis reached maximum values within 4 hours after dilution, with ornithine decarboxylase levels increasing lo-20 fold and nucleolar RNA synthesis increasing by about 60% during this period. 0.5 til putrescine effectively inhibited the rise in ornithine decarboxylase following cell transfer, but did not prevent increases in the rate of nucleolar RNA synthesis.
wnut eo ar RNA synthesis
In cultured
mammalian
nucleotide
incorporation
with
actual
the
presence
amino
this
the
acid
addition
deprivation acid
for
The mechanism
by which
RNA synthesis
is not
starvation,
into
this
response
'Abbreviations:
that FL,
assume that of the rate is
given
amino acid
starvation Grumnt
intracellular includes
Friend
0 1978
by Academic Press, Inc. in any form reserved.
of reproduction
in reference
3. the
proposed
rate
RNA.
Our
of nucleolar
that
of nucleolar
Decarboxylase.
of into
amino
of ATP and GTP,produces
ODC, Ornithine
1054
by 75% two hours
of C3Hluridine
in the rate
by
In FL cells,l
of that
and Grumnt
a decrease
0006-291X/78/0813~1054$01.00/0 Copyright All rights
caused
For the purposes
decreases
of
deprivation
of synthesis
levels
Leukemia;
(3).
rate
is seen both
inhibited
incorporation
assumption
by decreasing
pleiotypic
RNA is
the
effect
such as histidinol.
nucleolar
understood.
This
functional
to the culture
index
decreases
RNA (l-3).
analogue,
we shall
RNA is a valid
starvation
and with
of histidinol
justification
acid
nucleolar
of E3Hluridine
comnunication,
nucleolar
amino
into
of an amino
incorporation after
cells,
acid a RNA
8lOCHEMCAL
Vol.81,No.3,1978
synthesis
(2).
in FL cells
However,
following
An alternative ornithine
we failed
amino
mechanism
RNA synthesis.
of ornithine
to putrescine,
changes
in its
Several
lines
function
of evidence
as an initiation
and ODC activities various for
disappearance
treated
cells
affinity
for
Studies
with
fold
proliferate dilution rate
we also medium
many different
types
of stationary
phase
In this nucleolar
show that
nucleolar Experimental
with
the
paper,
and its
role
the decarboxylation 10 and 20 minutes
to respond
rapidly
of mammalian very
cultures
(4).
to
into
RNA about
we report
that, rates
of cells
inhibitors,
fresh
as expected,
and ODC levels
addition
of putrescine
at the time
increase
in ODC levels
but
lives
in cycloheximide and specific
indicate cells
that
a many-
are stimulated
to
to proliferate
medium,
two fold
with
2) the half
a strong
after
ODC may
1) RNA polymerase
stimulation
are stimulated
with
that
1 (5):
systems
early
cultures
RNA synthesis
to suogest
1 activities
3) ODC displays
occurs
When FL cell
blocks
during
treatment
1.
incorporation
able
RNA polymerase
correlated
(6,7).
(8).
increases
for
RNA polymerase
of uridfne
hours
of between
of OOC is
and
in ODC activity
catalyzes
Manen and Russell
during
identical,
on the enzyme
ET 4.1*1*17)
which
of ODC and RNA polymerase are
increase
led factor
and also
studies
or degradation.
are tightly
hormones
by recent
enzyme,
level
in ATP and GTP levels
(3).
has a half-life
of synthesis
decreases
carboxy-lyase,
This
intracellular rate
starvation
(L-ornithine
in nucleolar
the
acid
to observe
is suggested
decarboxylase
Therefore,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
they
during this
increase the
first
their few
stimulation
in FL cells. of dilution
does not prevent
by
However, into
the
fresh
increase
in
RNA synthesis. Procedures
Friend Leukemia cells [cell line 745(15), clone 18 obtained from %%vid Kabatl were gMn in Eagle's medium supplemented'wfth 10% fetal calf serum as previously described (9). Cells were diluted into fresh medium at a density of 5-7 x lo5 cells/ml 48 hours prior to use for experiments. Cells to be used for experiments were centrifuged. 3 minutes at room temperature at l/2 speed in an International Clinical centripge (Model CL). They were resuspended in fresh medium at a density of 1 x 10 cells/ml.
1055
1
BIOCHEMICAL
Vol. 81, No. 3, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ODC levels, nucleolar and nucleoplasmfc RNP synthesis rates at var ous trmes after resuspension in fresh medium in the presence and absence A. ODC specific activity. B. Nucleolar RNA synthesis of 0.5 mt-! putrescine. C. Nucleoplasmic RNA synthesis rate. Open circles, with putrescine; rate. closed circles, without putrescine. The error bars indicate the range of values obtained in two independent experiments; the lines are drawn through the average values. All values are given as percent of the zero time control.
Y-
Cell extracts were prepared the method of Prouty (10) was determined by the method'of
k%%%.. extracts
and ODC activity in extracts was Protein concentration of crude cell Bradford (11).
Nucleolar and nucleoplasmic Nucleolar and nucleoplasmic RNA synthesis rates: RNA synthesis rates were determined by measuring incorporation of r3Hluridine into nucleolar and nucleoplasmic fractions as previously described (3). Results
and Discussion
When stationary
phase
FL cells
that
permits
(1 x 106 per ml) approximately increases is
12 fold by about
with
added
to the cells
RNA synthesis
the
results
at the time
was completely was,
if
anything,
whereas
abolished,
which
When 0.5
whereas
greater
than
that
the
by
in Figure
ml1 putrescine
medium, increase
observed
rate
RNA synthesis
are shown
in fresh
density
RNA synthesis
Nucleoplasmic
(3,7).
of resuspension
increase
the nucleolar
results,
studies
medium at a cell
ODC levels
same period.
These
of earlier
in fresh
to resume,
4 hours
affected.
aqree
in ODC activity
growth
E5X during
not significantly the
within
are suspended
the
1, is
increase
in nucleolar
in the absence
of
putrescine. We have not established induction
whether
of ClOC in FL cells
the mechanism involves
requlation
1056
by which at the
putrescine translational
blocks lev1?1
Vol. 8 1, No. 3, 1978
(12)
or whether
8lOCHEMlCAL
it
involves
mechanism,
our observation
stimulation
of nucleolar
and Russell
that
However,
since
our results inhibition this
of the
rule
that
to ODC (14) This
of an nDr antizyme
ODC induction
RNA synthesis
activity nucleolar
could
resolve
for
rather
the possibility
than
that
RIJA synthesis.
work was supported
the proposal
impairing Studies
of Manen
1 (5).
the level
putrescine
the
from
RNA polymerase
of ODC without
this
Whatever
can be dissociated
factor
ODC activity,
out
(13).
does not support
an initiation
enzymatic
to stimulate
Acknowledqement:
induction
we measured
do not
protein
antibodies
fVlC is
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of ODC protein,
may cause the ability with
of
monospecific
question. by NIH grant
5 ROl CA16557.
References Hershko, A., Mamont, P., Shields, R., Tomkins, G. M. (1971) Nature New Biol. 323, 206-211. Grummt, F., Grumnt, I. (1976) Eur. J. Biochem. 64, 307-312. Dehlinger, P. J., Hamilton, T. A., Litt, M. (19rT) Eur. J. Biochem. 77, 495-499. Clark, J. L. (1974) Biochemistry 13, 4668-4674. Manen, C., Russell, D. H. (1977) Eience 195. 505-506. Acad. Sci., U.S.A. 60, Russell, D. H., Snyder, S. H. (1968) Procxat. 1420-1427. Tabor, H., Tabor, C. W. (1972) Adv. Enzymol. 36, 203-268. Sherton, C. C., Kabat, D. (1976) Dev. Biol. 4x 118-131. Hamilton, T. A., Litt, M. (1976) Biochim. Biophys. Acta 435, 362-375. Prouty, W. F. (1976) J. Cell Physiol. 89, 65-76. Bradford, M. M. (1976) Anal. Biochem. 7?!, 248-254. Clark, J. L., Fuller, J. L. (1975) Bioxemistry 2, 4403-4409. Heller, J. S., Fong, W. F., Cannelakis, E. S. (1976) Proc. Nat. Acad. Sci., U.S.A. 73, 1858-1862. nbenrader, ti. F.>routy, W. F. (1977) J. Biol. Chen. 252, 2866-2872. Friend, C. W., Scher, J. G., Holland, J. G., Sato, T. (1971) Proc. Nat. Acad. Sci., U.S.A. 68, 378-382.
1057
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
April 14,1978
Pages 1054-1057
ORNITHINE DECARBOXYLASE INDUCTION AND NUCLEOLAR RNA SYNTHESIS IN FRIEND LEUKEMIA CELLS P. J. Department
Dehlinger
of Biochemistry
and Division
School University
Received
March
and M. Litt of Medical
Genetics
of Medicine
of Oregon
Health
Sciences
Portland,
Oregon
97201
Center
1, 1978
The induction
of ornithine decarboxylase and the stimulation of following dilution of stationary phase Friend Leukemia Cells into fresh medium were studied. Ornithine decarboxylase activity and the rate of nucleolar RNA synthesis reached maximum values within 4 hours after dilution, with ornithine decarboxylase levels increasing lo-20 fold and nucleolar RNA synthesis increasing by about 60% during this period. 0.5 til putrescine effectively inhibited the rise in ornithine decarboxylase following cell transfer, but did not prevent increases in the rate of nucleolar RNA synthesis.
wnut eo ar RNA synthesis
In cultured
mammalian
nucleotide
incorporation
with
actual
the
presence
amino
this
the
acid
addition
deprivation acid
for
The mechanism
by which
RNA synthesis
is not
starvation,
into
this
response
'Abbreviations:
that FL,
assume that of the rate is
given
amino acid
starvation Grumnt
intracellular includes
Friend
0 1978
by Academic Press, Inc. in any form reserved.
of reproduction
in reference
3. the
proposed
rate
RNA.
Our
of nucleolar
that
of nucleolar
Decarboxylase.
of into
amino
of ATP and GTP,produces
ODC, Ornithine
1054
by 75% two hours
of C3Hluridine
in the rate
by
In FL cells,l
of that
and Grumnt
a decrease
0006-291X/78/0813~1054$01.00/0 Copyright All rights
caused
For the purposes
decreases
of
deprivation
of synthesis
levels
Leukemia;
(3).
rate
is seen both
inhibited
incorporation
assumption
by decreasing
pleiotypic
RNA is
the
effect
such as histidinol.
nucleolar
understood.
This
functional
to the culture
index
decreases
RNA (l-3).
analogue,
we shall
RNA is a valid
starvation
and with
of histidinol
justification
acid
nucleolar
of E3Hluridine
comnunication,
nucleolar
amino
into
of an amino
incorporation after
cells,
acid a RNA
8lOCHEMCAL
Vol.81,No.3,1978
synthesis
(2).
in FL cells
However,
following
An alternative ornithine
we failed
amino
mechanism
RNA synthesis.
of ornithine
to putrescine,
changes
in its
Several
lines
function
of evidence
as an initiation
and ODC activities various for
disappearance
treated
cells
affinity
for
Studies
with
fold
proliferate dilution rate
we also medium
many different
types
of stationary
phase
In this nucleolar
show that
nucleolar Experimental
with
the
paper,
and its
role
the decarboxylation 10 and 20 minutes
to respond
rapidly
of mammalian very
cultures
(4).
to
into
RNA about
we report
that, rates
of cells
inhibitors,
fresh
as expected,
and ODC levels
addition
of putrescine
at the time
increase
in ODC levels
but
lives
in cycloheximide and specific
indicate cells
that
a many-
are stimulated
to
to proliferate
medium,
two fold
with
2) the half
a strong
after
ODC may
1) RNA polymerase
stimulation
are stimulated
with
that
1 (5):
systems
early
cultures
RNA synthesis
to suogest
1 activities
3) ODC displays
occurs
When FL cell
blocks
during
treatment
1.
incorporation
able
RNA polymerase
correlated
(6,7).
(8).
increases
for
RNA polymerase
of uridfne
hours
of between
of OOC is
and
in ODC activity
catalyzes
Manen and Russell
during
identical,
on the enzyme
ET 4.1*1*17)
which
of ODC and RNA polymerase are
increase
led factor
and also
studies
or degradation.
are tightly
hormones
by recent
enzyme,
level
in ATP and GTP levels
(3).
has a half-life
of synthesis
decreases
carboxy-lyase,
This
intracellular rate
starvation
(L-ornithine
in nucleolar
the
acid
to observe
is suggested
decarboxylase
Therefore,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
they
during this
increase the
first
their few
stimulation
in FL cells. of dilution
does not prevent
by
However, into
the
fresh
increase
in
RNA synthesis. Procedures
Friend Leukemia cells [cell line 745(15), clone 18 obtained from %%vid Kabatl were gMn in Eagle's medium supplemented'wfth 10% fetal calf serum as previously described (9). Cells were diluted into fresh medium at a density of 5-7 x lo5 cells/ml 48 hours prior to use for experiments. Cells to be used for experiments were centrifuged. 3 minutes at room temperature at l/2 speed in an International Clinical centripge (Model CL). They were resuspended in fresh medium at a density of 1 x 10 cells/ml.
1055
1
BIOCHEMICAL
Vol. 81, No. 3, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ODC levels, nucleolar and nucleoplasmfc RNP synthesis rates at var ous trmes after resuspension in fresh medium in the presence and absence A. ODC specific activity. B. Nucleolar RNA synthesis of 0.5 mt-! putrescine. C. Nucleoplasmic RNA synthesis rate. Open circles, with putrescine; rate. closed circles, without putrescine. The error bars indicate the range of values obtained in two independent experiments; the lines are drawn through the average values. All values are given as percent of the zero time control.
Y-
Cell extracts were prepared the method of Prouty (10) was determined by the method'of
k%%%.. extracts
and ODC activity in extracts was Protein concentration of crude cell Bradford (11).
Nucleolar and nucleoplasmic Nucleolar and nucleoplasmic RNA synthesis rates: RNA synthesis rates were determined by measuring incorporation of r3Hluridine into nucleolar and nucleoplasmic fractions as previously described (3). Results
and Discussion
When stationary
phase
FL cells
that
permits
(1 x 106 per ml) approximately increases is
12 fold by about
with
added
to the cells
RNA synthesis
the
results
at the time
was completely was,
if
anything,
whereas
abolished,
which
When 0.5
whereas
greater
than
that
the
by
in Figure
ml1 putrescine
medium, increase
observed
rate
RNA synthesis
are shown
in fresh
density
RNA synthesis
Nucleoplasmic
(3,7).
of resuspension
increase
the nucleolar
results,
studies
medium at a cell
ODC levels
same period.
These
of earlier
in fresh
to resume,
4 hours
affected.
aqree
in ODC activity
growth
E5X during
not significantly the
within
are suspended
the
1, is
increase
in nucleolar
in the absence
of
putrescine. We have not established induction
whether
of ClOC in FL cells
the mechanism involves
requlation
1056
by which at the
putrescine translational
blocks lev1?1
Vol. 8 1, No. 3, 1978
(12)
or whether
8lOCHEMlCAL
it
involves
mechanism,
our observation
stimulation
of nucleolar
and Russell
that
However,
since
our results inhibition this
of the
rule
that
to ODC (14) This
of an nDr antizyme
ODC induction
RNA synthesis
activity nucleolar
could
resolve
for
rather
the possibility
than
that
RIJA synthesis.
work was supported
the proposal
impairing Studies
of Manen
1 (5).
the level
putrescine
the
from
RNA polymerase
of ODC without
this
Whatever
can be dissociated
factor
ODC activity,
out
(13).
does not support
an initiation
enzymatic
to stimulate
Acknowledqement:
induction
we measured
do not
protein
antibodies
fVlC is
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of ODC protein,
may cause the ability with
of
monospecific
question. by NIH grant
5 ROl CA16557.
References Hershko, A., Mamont, P., Shields, R., Tomkins, G. M. (1971) Nature New Biol. 323, 206-211. Grummt, F., Grumnt, I. (1976) Eur. J. Biochem. 64, 307-312. Dehlinger, P. J., Hamilton, T. A., Litt, M. (19rT) Eur. J. Biochem. 77, 495-499. Clark, J. L. (1974) Biochemistry 13, 4668-4674. Manen, C., Russell, D. H. (1977) Eience 195. 505-506. Acad. Sci., U.S.A. 60, Russell, D. H., Snyder, S. H. (1968) Procxat. 1420-1427. Tabor, H., Tabor, C. W. (1972) Adv. Enzymol. 36, 203-268. Sherton, C. C., Kabat, D. (1976) Dev. Biol. 4x 118-131. Hamilton, T. A., Litt, M. (1976) Biochim. Biophys. Acta 435, 362-375. Prouty, W. F. (1976) J. Cell Physiol. 89, 65-76. Bradford, M. M. (1976) Anal. Biochem. 7?!, 248-254. Clark, J. L., Fuller, J. L. (1975) Bioxemistry 2, 4403-4409. Heller, J. S., Fong, W. F., Cannelakis, E. S. (1976) Proc. Nat. Acad. Sci., U.S.A. 73, 1858-1862. nbenrader, ti. F.>routy, W. F. (1977) J. Biol. Chen. 252, 2866-2872. Friend, C. W., Scher, J. G., Holland, J. G., Sato, T. (1971) Proc. Nat. Acad. Sci., U.S.A. 68, 378-382.
1057
