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Effect of hydroxyurea on regenerating rat liver
Vol. 19, No. 5, 1965
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EFFECT OF HYDROXYUREA ON REGENERATING RAT LIVER * John W. Yarbro,
W. G. Niehaus**,
and Cyrus
P. Barnum
Departments of Medicine and Biochemistry, University of Minnesota College of Medical Sciences, Minneapolis, Minnesota
ReceivedApril5,
1965
Hydroxyurea synthesis
in HeLa cells
bacteria is
(14).
with
there
is
synthesized
during
two processes
has not
synthesis
of isotope
MATERIALS
its
and mode of action
to deoxyribo-
formation
of dCMP when CMP
from rats have been
rat
indicating
previously
observed
that
treated
on synthesis
of
the cell
do not produce
cycle
interdependence
of these
Doses of radiation immediate
sufficient
inhibition
of
(12,16). the interdependence
we studied into
the effect
of the synthesis
of hydroxyurea
DNA, RNA, and several
AND METHODS:
on the incorpo-
protein
fractions
Male
Holtzman
rats,
weighing
of
300 to 350 grams
__-_________-__________
Presently, Chemical
of
liver.
* This research was supported in part Research Grant No. CA-01747-13. **
of
DNA and nuclear
the same period
been resolved.
to investigate
DNA and histone,
regenerating
seen in
of the possible
DNA synthesis
In order
ration
is
of DNA
(15),
of ribonucleotides
evidence
the question
histone
cells
has been made that
No effects
(1,2,6,9,13),
to inhibit
inhibitor
(17,14).
Although are
tumor
preparations
(5).
RNA or protein
to be a potent
ascites
inhibition
to bone marrow
hydroxyurea
histone
(17),
of conversion
since
added
shown
The suggestion
at the level
nucleotides is
has been
post-doctoral Engineering,
Fellow, University
592
by Public
Department of Illinois,
Health
Service
of Chemistry and Urbana, Illinois
Vol. 19, No. 5, 1965
were
used.
Higgins All
BIOCHEMICAL
Partial
hepatectomies
and Anderson
partial
later,
(7)
hepatectcnnies
1 ml of physiological
later,
animals
received
saline
by the method
by Brues,
performed
Drury,
at 8 AM.
the isotope
was administered
32P as sodium
Twenty-four
hours
(6.75
Inc.,
mc/mmole,
in
minutes
manner:
(Iso-Serve
(3).
control
Thirty
in a similar
or 25 uc of glycine-1-14C
and Brues
injection;
saline.
phosphate
of
100 mg of hydroxyurea
by intraperitoneal
1 ml of physiological
free
performed
were
received
carrier Mass.)
were
as modified
the experimental
animals
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
500 nc of
Cambridge,
Tracerlab,
Waltham,
Mass.). One hour and their
after
livers
injection
of isotope
homogenized
in
ice
DNA, RNA, and acid-soluble specific
activities
A portion 105,000 supernatant hot
fluid
was dissolved
referred
in dilute
to as cytoplasmic
Nuclei Chauveau,
were
extracted
with
0.25
Simson,
purification
was dissolved
basic
proteins The nuclear
had been extracted
(10). at
After
from
This
the
extraction
ethanol-ether,
NaOH.
(4).
and Butler
although are
hot
with
and ether,
protein
the
is hereafter
it
nuclear Histone
nuclei proteins
was purified
The final
(8).
in 0.05 is
of the method
The isolated
N H2SO4.
of Laurence,
to as histone
and their
was precipitated acid.
and the acid-soluble
cold
described
by a modification
and Rouiller
0.25 M sucrose
isolated
protein.
isolated
Mould,
sacrificed
was centrifuged
Protein
ethanol,
were
M sucrose.
were
homogenate
trichloroacetic
acid,
0.25
as previously
minutes.
with
trichloroacetic
protein
in
of the initial sixty
cold
nucleotides
determined
x g for
the animals
product
of
were by the method of this
N HCl and is hereafter
recognized
that
small
washed
were
referred
amounts
of other
present. sediment
from which
was treated
with
593
the buffered
acid-soluble
nuclear
protein
10% NaCl at 1000 to
Vol. 19, No. 5, 1965
solubilize
BIOCHEMICAL
the nucleic
5% trichloroacetic That
portion
referred
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
acids
acid,
(10)
ethanol,
of the residue
previously
activities described
HESDLTS:
Table
to that
extracted
ethanol-ether,
dissolved
nuclear
with
hot
and ether.
in dilute
NaOH is
protein.
of the protein
samples
were
determined
the specific
of the acid-soluble
into
DNA is markedly
ration
of 32P into
activity
nucleotides.
reduced
with
of RNA and DNA relative Incorporation
respect
of isotope
to the control;
RNA is not measurably
incorpo-
affected.
Effect of Hydroxyurea on Isotope Incorporation into DNA and RNA. (Specific specific
activity, activity
Control
c/m/rig P, shown as per cent of of acid-soluble nucleotide phosphorus)
Animals
Hydroxyurea
Treated
Animals
DNAP*
RNAP*
DNAP*
RNAP*
1.38 1.47 1.75 1.81 2.08 2.34 2.58 3.16
2.23 3.36 2.64 2.61 2.54 2.10 2.75 2.64
0.26 0.41 0.48 0.51 0.55 0.73 0.75
2.88 2.60 2.71 2.91 2.35 2.36 2.41
2.07 +- 0.21 (Mean + S.E.)
2.61
*DNAP and HNAP denote
Table fractions. treated of glycine protein. animals
as
(9).
I shows
TABLE1
hot
which
to as acid-insoluble
Specific
and was then
II
shows There
+_ 0.13
0.53
DNA phosphorus
the specific
and the control
group
into
the cytoplasmic
protein
in
with
the case of histone,
show a mean value
which
of the three
difference
animals
is only
594
2.61 f 0.04
and HNA phosphorus
activity
is no significant
However,
+- 0.06
protein
between respect
to incorporation
and acid-insoluble the hydroxyurea 61% of that
the hydroxyurea
nuclear treated
of the control
Vol. 19, No. 5, 1965
group.
BIOCHEMICAL
The difference
AND BIOPHYSICAL RESEARCH COMMUNICATiONS
of these
means is
significant
at a level
of
P = 0.02. TABLE II
Effect
of Hydroxyurea on Isotope Incorporation into Several Protein Fractions
(Specific
activity
expressed Control
Histone
3450 3080 3620 4680 3900 4210
6180 5310 5620 8000 5610 5740
3820 +- 240
6080 +- 390
Hydroxyurea
Barnum
5070 6090 5430 5040 7110 8000
2310 +_ 410
3510 + 170
6120 + 500
The finding DNA with
with
that
for
no effect (17)
with
HeLa cells
tumor
by Niehaus
and Barnum
total
DNA (11)
inhibits
on RNA is
two fractions
in accord and Yarbro,
The magnitude
cells.
of isolated (10)
did
not
nuclei
was found
and the incorporation
fractions
the incorporation
appear
with
the results
Kennedy
and
of inhibition prepared
as
to be comparable of the isotope
to be affected
to into
by
(11).
The finding of isotope
hydroxyurea
in
the RNA of these hydroxyurea
that
ascites
of DNA synthesis described
Animals
3170 3360 3340 3090 4210 3880
and Hodas (15)
Treated
1290 1640 1890 2080 3100 3880
DISCUSSION:
of Young
Acid-insoluble nuclear protein
protein
3800 + 310 (Mean +_ S.E.)
protein)
Animals
Cytoplasmic
2940 3220 3220 4160 4380 4860
of 32P into
as c/m/mg
into
that
hydroxyurea
cytoplasmic
does not
protein
or into
595
inhibit nuclear
the incorporation acid-insoluble
Vol. 19, No. 5, 1965
protein
BIOCHEMICAL
is
in accord
no inhibition the work
synthesis
hydroxyurea
which
previously
workers
synthesized
(6)
histone
molecules.
and with of
of concentrations
incorporation
an inhibition
into
of
nuclear
of DNA synthesis,
involving
synthesis
erating
rat
that
has not
nucleoprotein
with
a portion
is dependent
SUMMARY:
Hydroxyurea
synthesis
in regenerating No detectable
14 C-glycine
into
rat
shown liver
difference
the incorporation
into
suggesting in
is a unitary
of DNA and
such an hypothesis, of nuclear
histone
addition
to be a potent
suggesting in regen-
that to,
or into nuclear
inhibitor
no measurable
was noted
protein
of histone
(1,2,6,9,13)
synthesis
duplication
with
cytoplasmic
value,
cycle
on DNA synthesis.
has been
synthesis.
and RNA are
of the cell
consistent
of at least
of the control
histone
the simultaneous
are
liver
but
that
the same period
Our findings
effect
cultures,
DNA synthesis.
have reported
during
process
synthesis
the presence
of isotope
has been suggested
protein,
who found
who found no inhibition
inhibited
with
(17)
been reported.
Various
that
in
inhibition
and Hodas
in HeLa cell
and Levy (14)
markedly
concomitant
of Young
synthesis
in bacteria
The specific histone,
the work
of protein of Rosenkranz
protein
and it
with
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
effect
of DNA on RNA
in incorporation acid-insoluble
histone
hydroxyurea
nuclear
was only may inhibit
or as a consequence
of
of,
61% the its
on DNA.
REFERENCES : 1. 2. 3. 4.
Alfert, M., Exptl. Cell Research, Suppl. s 227 (1958). Bloch, D. P., J. Cellular Comp. Physiol. 3 Suppl. & 87 (1963). Brues; A. M., Drury, D. R. and Brues, M.C., Arch. Pathol. 658 (1936). Chauveau, J;, MO&, Y. and Rouiller, C., Exptl. Cell Research 5317 (1956).
596
3
Vol. 19, No. 5, 1965
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Frenkel, E. P., Skinner, W. N., and Smiley, J. P., Cancer Chemotherapy Reports 3 19 (1964). Gall, J. G., J. Biophys. Biochem. Cytol. 2 295 (1959). Higgins, G. M. and Anderson, R.M., Arch. Pathol. I& 186 (1931). Laurence, D. J. R., Simson, P. and Butler, J. A. V., Biochem. J. (London) a 200 (1963). Niehaus, W. G. and Barnum, C. P., Bxptl. Cell Research, in press. Niehaus, W. G. and Barnum, C. P., J. Biol. Chem. 239. 1198 (1964). observations. Niehaus, W. G., unpublished Ontoko,.J. andMoorehead, W., Biochim. et Biophys. Acta s 685 (1964). Prescott, D. M. and Kimball, R. F., Proc. Natl. Acad. Sci. U.S. 47- 686 (1961). Rosenkranz, H. S. and Levy, J. A., Biochim. et Biophys. Acta 95- 181 (1965). Yarbro, J. W., Kennedy, B. J. and Barnum, C. P., Proc. Natl. Acad. Sci. U.S., in press. Yarbro, J. W. and Barnum, C. P., unpublished observations. Young, C. W. and Hodas, S., Science 146, 1172 (1964).
597
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EFFECT OF HYDROXYUREA ON REGENERATING RAT LIVER * John W. Yarbro,
W. G. Niehaus**,
and Cyrus
P. Barnum
Departments of Medicine and Biochemistry, University of Minnesota College of Medical Sciences, Minneapolis, Minnesota
ReceivedApril5,
1965
Hydroxyurea synthesis
in HeLa cells
bacteria is
(14).
with
there
is
synthesized
during
two processes
has not
synthesis
of isotope
MATERIALS
its
and mode of action
to deoxyribo-
formation
of dCMP when CMP
from rats have been
rat
indicating
previously
observed
that
treated
on synthesis
of
the cell
do not produce
cycle
interdependence
of these
Doses of radiation immediate
sufficient
inhibition
of
(12,16). the interdependence
we studied into
the effect
of the synthesis
of hydroxyurea
DNA, RNA, and several
AND METHODS:
on the incorpo-
protein
fractions
Male
Holtzman
rats,
weighing
of
300 to 350 grams
__-_________-__________
Presently, Chemical
of
liver.
* This research was supported in part Research Grant No. CA-01747-13. **
of
DNA and nuclear
the same period
been resolved.
to investigate
DNA and histone,
regenerating
seen in
of the possible
DNA synthesis
In order
ration
is
of DNA
(15),
of ribonucleotides
evidence
the question
histone
cells
has been made that
No effects
(1,2,6,9,13),
to inhibit
inhibitor
(17,14).
Although are
tumor
preparations
(5).
RNA or protein
to be a potent
ascites
inhibition
to bone marrow
hydroxyurea
histone
(17),
of conversion
since
added
shown
The suggestion
at the level
nucleotides is
has been
post-doctoral Engineering,
Fellow, University
592
by Public
Department of Illinois,
Health
Service
of Chemistry and Urbana, Illinois
Vol. 19, No. 5, 1965
were
used.
Higgins All
BIOCHEMICAL
Partial
hepatectomies
and Anderson
partial
later,
(7)
hepatectcnnies
1 ml of physiological
later,
animals
received
saline
by the method
by Brues,
performed
Drury,
at 8 AM.
the isotope
was administered
32P as sodium
Twenty-four
hours
(6.75
Inc.,
mc/mmole,
in
minutes
manner:
(Iso-Serve
(3).
control
Thirty
in a similar
or 25 uc of glycine-1-14C
and Brues
injection;
saline.
phosphate
of
100 mg of hydroxyurea
by intraperitoneal
1 ml of physiological
free
performed
were
received
carrier Mass.)
were
as modified
the experimental
animals
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
500 nc of
Cambridge,
Tracerlab,
Waltham,
Mass.). One hour and their
after
livers
injection
of isotope
homogenized
in
ice
DNA, RNA, and acid-soluble specific
activities
A portion 105,000 supernatant hot
fluid
was dissolved
referred
in dilute
to as cytoplasmic
Nuclei Chauveau,
were
extracted
with
0.25
Simson,
purification
was dissolved
basic
proteins The nuclear
had been extracted
(10). at
After
from
This
the
extraction
ethanol-ether,
NaOH.
(4).
and Butler
although are
hot
with
and ether,
protein
the
is hereafter
it
nuclear Histone
nuclei proteins
was purified
The final
(8).
in 0.05 is
of the method
The isolated
N H2SO4.
of Laurence,
to as histone
and their
was precipitated acid.
and the acid-soluble
cold
described
by a modification
and Rouiller
0.25 M sucrose
isolated
protein.
isolated
Mould,
sacrificed
was centrifuged
Protein
ethanol,
were
M sucrose.
were
homogenate
trichloroacetic
acid,
0.25
as previously
minutes.
with
trichloroacetic
protein
in
of the initial sixty
cold
nucleotides
determined
x g for
the animals
product
of
were by the method of this
N HCl and is hereafter
recognized
that
small
washed
were
referred
amounts
of other
present. sediment
from which
was treated
with
593
the buffered
acid-soluble
nuclear
protein
10% NaCl at 1000 to
Vol. 19, No. 5, 1965
solubilize
BIOCHEMICAL
the nucleic
5% trichloroacetic That
portion
referred
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
acids
acid,
(10)
ethanol,
of the residue
previously
activities described
HESDLTS:
Table
to that
extracted
ethanol-ether,
dissolved
nuclear
with
hot
and ether.
in dilute
NaOH is
protein.
of the protein
samples
were
determined
the specific
of the acid-soluble
into
DNA is markedly
ration
of 32P into
activity
nucleotides.
reduced
with
of RNA and DNA relative Incorporation
respect
of isotope
to the control;
RNA is not measurably
incorpo-
affected.
Effect of Hydroxyurea on Isotope Incorporation into DNA and RNA. (Specific specific
activity, activity
Control
c/m/rig P, shown as per cent of of acid-soluble nucleotide phosphorus)
Animals
Hydroxyurea
Treated
Animals
DNAP*
RNAP*
DNAP*
RNAP*
1.38 1.47 1.75 1.81 2.08 2.34 2.58 3.16
2.23 3.36 2.64 2.61 2.54 2.10 2.75 2.64
0.26 0.41 0.48 0.51 0.55 0.73 0.75
2.88 2.60 2.71 2.91 2.35 2.36 2.41
2.07 +- 0.21 (Mean + S.E.)
2.61
*DNAP and HNAP denote
Table fractions. treated of glycine protein. animals
as
(9).
I shows
TABLE1
hot
which
to as acid-insoluble
Specific
and was then
II
shows There
+_ 0.13
0.53
DNA phosphorus
the specific
and the control
group
into
the cytoplasmic
protein
in
with
the case of histone,
show a mean value
which
of the three
difference
animals
is only
594
2.61 f 0.04
and HNA phosphorus
activity
is no significant
However,
+- 0.06
protein
between respect
to incorporation
and acid-insoluble the hydroxyurea 61% of that
the hydroxyurea
nuclear treated
of the control
Vol. 19, No. 5, 1965
group.
BIOCHEMICAL
The difference
AND BIOPHYSICAL RESEARCH COMMUNICATiONS
of these
means is
significant
at a level
of
P = 0.02. TABLE II
Effect
of Hydroxyurea on Isotope Incorporation into Several Protein Fractions
(Specific
activity
expressed Control
Histone
3450 3080 3620 4680 3900 4210
6180 5310 5620 8000 5610 5740
3820 +- 240
6080 +- 390
Hydroxyurea
Barnum
5070 6090 5430 5040 7110 8000
2310 +_ 410
3510 + 170
6120 + 500
The finding DNA with
with
that
for
no effect (17)
with
HeLa cells
tumor
by Niehaus
and Barnum
total
DNA (11)
inhibits
on RNA is
two fractions
in accord and Yarbro,
The magnitude
cells.
of isolated (10)
did
not
nuclei
was found
and the incorporation
fractions
the incorporation
appear
with
the results
Kennedy
and
of inhibition prepared
as
to be comparable of the isotope
to be affected
to into
by
(11).
The finding of isotope
hydroxyurea
in
the RNA of these hydroxyurea
that
ascites
of DNA synthesis described
Animals
3170 3360 3340 3090 4210 3880
and Hodas (15)
Treated
1290 1640 1890 2080 3100 3880
DISCUSSION:
of Young
Acid-insoluble nuclear protein
protein
3800 + 310 (Mean +_ S.E.)
protein)
Animals
Cytoplasmic
2940 3220 3220 4160 4380 4860
of 32P into
as c/m/mg
into
that
hydroxyurea
cytoplasmic
does not
protein
or into
595
inhibit nuclear
the incorporation acid-insoluble
Vol. 19, No. 5, 1965
protein
BIOCHEMICAL
is
in accord
no inhibition the work
synthesis
hydroxyurea
which
previously
workers
synthesized
(6)
histone
molecules.
and with of
of concentrations
incorporation
an inhibition
into
of
nuclear
of DNA synthesis,
involving
synthesis
erating
rat
that
has not
nucleoprotein
with
a portion
is dependent
SUMMARY:
Hydroxyurea
synthesis
in regenerating No detectable
14 C-glycine
into
rat
shown liver
difference
the incorporation
into
suggesting in
is a unitary
of DNA and
such an hypothesis, of nuclear
histone
addition
to be a potent
suggesting in regen-
that to,
or into nuclear
inhibitor
no measurable
was noted
protein
of histone
(1,2,6,9,13)
synthesis
duplication
with
cytoplasmic
value,
cycle
on DNA synthesis.
has been
synthesis.
and RNA are
of the cell
consistent
of at least
of the control
histone
the simultaneous
are
liver
but
that
the same period
Our findings
effect
cultures,
DNA synthesis.
have reported
during
process
synthesis
the presence
of isotope
has been suggested
protein,
who found
who found no inhibition
inhibited
with
(17)
been reported.
Various
that
in
inhibition
and Hodas
in HeLa cell
and Levy (14)
markedly
concomitant
of Young
synthesis
in bacteria
The specific histone,
the work
of protein of Rosenkranz
protein
and it
with
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
effect
of DNA on RNA
in incorporation acid-insoluble
histone
hydroxyurea
nuclear
was only may inhibit
or as a consequence
of
of,
61% the its
on DNA.
REFERENCES : 1. 2. 3. 4.
Alfert, M., Exptl. Cell Research, Suppl. s 227 (1958). Bloch, D. P., J. Cellular Comp. Physiol. 3 Suppl. & 87 (1963). Brues; A. M., Drury, D. R. and Brues, M.C., Arch. Pathol. 658 (1936). Chauveau, J;, MO&, Y. and Rouiller, C., Exptl. Cell Research 5317 (1956).
596
3
Vol. 19, No. 5, 1965
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Frenkel, E. P., Skinner, W. N., and Smiley, J. P., Cancer Chemotherapy Reports 3 19 (1964). Gall, J. G., J. Biophys. Biochem. Cytol. 2 295 (1959). Higgins, G. M. and Anderson, R.M., Arch. Pathol. I& 186 (1931). Laurence, D. J. R., Simson, P. and Butler, J. A. V., Biochem. J. (London) a 200 (1963). Niehaus, W. G. and Barnum, C. P., Bxptl. Cell Research, in press. Niehaus, W. G. and Barnum, C. P., J. Biol. Chem. 239. 1198 (1964). observations. Niehaus, W. G., unpublished Ontoko,.J. andMoorehead, W., Biochim. et Biophys. Acta s 685 (1964). Prescott, D. M. and Kimball, R. F., Proc. Natl. Acad. Sci. U.S. 47- 686 (1961). Rosenkranz, H. S. and Levy, J. A., Biochim. et Biophys. Acta 95- 181 (1965). Yarbro, J. W., Kennedy, B. J. and Barnum, C. P., Proc. Natl. Acad. Sci. U.S., in press. Yarbro, J. W. and Barnum, C. P., unpublished observations. Young, C. W. and Hodas, S., Science 146, 1172 (1964).
597