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490

ORN VP-490
Third International Symposium
Regulation of Enzyme Activity and Synthesis
in Normal and Neoplastic Tissues"
October 5, 6 1964
Indiana University School of Medicino
Indianapolis, Indiana

MASTER
RNA Synthesis and Enzyme Induction by Hydrocortisoned
F.1. Kenney, D. L. Greenman, W. D. wicka' and W. L. Albritton
Biology Division
Oak Ridge National Laboratory
Oak Ridge, Tennesson
coparend
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Research sponsored jointly by the United States Atomic Energy Commission
under contract with the Union Carbide Corporation and the National Cancer In-
stitute, National Institutes of Health.
2National Cancer Institute Postdoctoral Fellow (Grant No. 6f2-CA-16,375-OLA!)
Present address: McCollum-Pratt Institute, The Johns Hopkins University,
Baltimore Maryland.
3American Cancer Society Postdoctoral Fellow
4Present address: University of Alabama Medical School, Birmingham, Alabama
Introduction
Enzyme induction in liver - when Induction 18 detined as an increase
in enzyme level - can be brought about by a variety of mechanisms, as 18
amply demonstrated elsewhere in this and in previous volumes of the Advances
in Enzyme Regulation serios. In some instances of Induction the mechanism
whereby an enzyme level is increased is known, and such is the case in
the inductions initiated by glucocorticoid hormones. Inductions of tyrosine
transaminase, (1) glutamic-alanine transaminase (2) and tryptophane pyrrolase(3, 4)
by glucorticoid hormones have now been conclusively demonstrated to be
due to an increased rate of enzyme synthesis. Thus analysis of the mechanism
of hormonal Induction can, in these instances, properly advance to the ques-
tion: how do adrenal steroids increase the rate of synthesis of specific
proteins? This question has led us to an analysis of hormonal effects on
RNA metabolism, and the results of our investigations in this area are
described below. These experiments have led, in turn, to a rephrasing of
the question asked above, which we now choose to pose as: do adrenal
steroids increase the rate of synthesis of specific proteins?
.
Results and Discussion
Any consideration of the possible mechanisms by which steroid
hormones may influence hepatic enzyme synthesis must take into account
*
.* *
.
.
.
.
*
:
* :*
the rapidity with which these hormones are removed from the liver. In
:*
.
:.
'
.-
.'
:.:
:
Figure 1 data are presented which demonstrate that hydrocortisone-induced
:
F-
:
:
enzyme synthesis reaches its maximal rate only after more than 90% of an
2
*
, ,..
.
,
-r..
i
administered dose of "c-labeled steroid has been removed from the liver.
While the possibility that the residual steroid may represent a particularly
active form cannot be excluded, this result suggests that the primary hor-
monal effect involves cellular events which procedo the actual formation
of the polypeptide enzyme. Attention is thereby directed towards the nu-
F-2
cleic acid components, thought to be regulating enzyme synthes 18.
Felgelson, Gross and Felgelson found that the turnover of RNA wus
Increased in all the subcellular fractions of liver, following a single dose
of cortisone.(5) By employing briet periods of exposure to 32p ("pulse-
labeling" ) we later found (y that the steroid effect is actually limited to
nuclear RNA synthesis, which is dramatically increased by the hormone,
while synthesis of cytoplasmic RNA was unchanged (Figure 2). Increased
synthesis of nuclear RNA precedes accumulation of the induced transaminase,
a result kinetically consistent with the conclusion that the primary hormonal
effect is on nuclear RNA synthesis, with the hormonally induced increase in
this parameter resulting, in turn, in increased enzyme synthesis. Also con-
sistent with this conclusion is the demonstration by Greengard and Acs()
that actinomycin treatment prevents the hormonal elevation of transaminase
activity, thus implicating DNA-directed RNA synthesis in this induction.
When longer periods of exposure to up were employed, an hormonal effect
on cytoplasmic RNA labeling bocomos apparent (Figure 3), in agreement
with the results of Foigelson et al.(5) The sequence of events following
hormone administration thus appears to be: (1) an increase in the rate of
nuclear RNA synthesis, (2) passage of the newly synthesized RNA into the
F-3
cytoplasın, and ( 3 ) utilization of this KNA In enzyme synthesis.
What is the functional nature of the KNA synthesized in response to
hydrocortisone? In attempting to answer this question we were fortunate
that research in several laboratories has been directed toward learning more
of the nature of the rapidly-labeled RNA of animal tissues and cells. From
these studies it is clear that the bulk of the rapidly-labeled RNA 18 made
up of two kinds of RNA. The first of these (PRNA) 18 nuclear in origin, 18)
is of high molecular weight, 18-11) has a boso composition like that of rRNA, (-)
and under appropriate experimental conditions can be shown to move into
the typical 28 and 18 8 (Svedberg units ) of rRNA.''.wine! These charac-
teristics identify pRNA as a precursor form of rRNA. In addition to PRNA
there is a second component ( dRNA ) which is synthesized in non-molecular
regions of the nucleus, (•) is quite heterogeneous in size, (8-12) has a base
composition like the cell :)NA, C-1) and which preferentially hybridizes with
DNA.(13) These characteristics Identify this RNA as dRNA, and it is possible
that this is the cellular mRNA. However positive identification of an RNA as
RNA must involve demonstrations of messenger activity, 1.e., the capacity
to code for the synthesis of a particular polypeptide chain must be shown.
While there have been numerous demonstrations of capacity to stimulate
amino acid incorporation into protein in cell-free systems there is as yet
Abbreviations employed are: PRNA, nuclear precursor to ribosomal
RNA; DRNA, DNA-Ilke RNA; mRNA, messenger RNA; rRNA, ribosomal RNA;
and tRNA, transfer RNA.
no firm proof of ability to code for the synthesis of a particular protuin.
Honce for the moment a positive functional designation of this RNA cannot
be made, and we therefore profer the operational designation dRNA.
Analysis of the base composition of the rapidly-labeled nuclear RNA,
and the finding that hormonal stimulation failed to alter this composition
I-
(Table 1) provided the first indication that synthesis of more than one
kind of RNA 16 Involved in hormonal enzyme Induction. Composition of
the newly-synthesized RNA from the nuclei of adrenalectomized animals
is like that of the total ( unlabeled ) nuclear RNA, and 18 Indicative of a
mixture of PRNA and dRNA. This result 18 in accord with the studies dis-
cussed above. Treatraent with hydrocortisone falled to alter this composition,
although the hormone effected a 2- to 3-fold increase in the rate of labeling
of RNA. We infer, then, that the hormonally-induced RNA must be, like
that synthesized in the absence of adrenal steroid, a mixture of pRNA and
dRNA.
Direct evidence for this conclusion was sought, employing a slight
modification of the thermal fractionation procedure introduced by Georgiev
and his collaborators. By this technique the ºp-labeled nuclear RNA
was separated into four fractions, containing RNA species differing in base
composition and sedimentation characteristics (Table 2). Fraction con- T-2
tains only low molecular weight 32p-RNA and will be discussed separately
below. Fractions 2, 3 and 4 contain the bulk of the RNA that becomes labeled
in a brief exposure to *p. While our experience with this technique has
not yielded the clear-cut separations of PRNA and dRNA reported by Georgiev et al.,(!)
the base composition c: Fraction 2 approaches that of rRNA, while the com-
position of Fraction 4 approaches that of dRNA. From these differences in
composition we have calculated the amounts of the two types of RNA present
in gach fraction. Hormone treatment did not appreciably change the results
of this fractionation, and it was therefore possible to determine whether or
not hydrocortisone effects a seleciive increase in one or the other types of
RNA. The results ( Table 3) show clearly that the hormone offect 18 equiva-
T-3
Jeni in all fractions, and thus we conclude that synthesis of both pRNA and
dRNA is stimulated by the hormone.
Whether tRNA synthesis is affected proved more difficult to resolve.
l'he: 32p-RNA recovered in Fraction 1 of the differential phenol extraction
sedimented with a sharp peak over the 4 S region known to contain tRNA.
Liver cytoplasm similarly contains P-RNA that sediments like tRNA. How-
ever, as we have shown (14) most of this 32p-RNA is not tRNA, but a mixture
of low molecular weight spocles that probably reflect degradation of pRNA
and/or dRNA. When tRNA is separated from this mixture by salt fractionation
( Table 4) or by DEAE chromatography, it is clear that most of the 32p.
labeling of tRNA is due to turnover of the -pCpCpA terminus of the rucleo-
1-4
tide chain. Labeling of the salt-insoluble RNA reflects de novo synthesis,
and the synthesis of this RNA was affected by the hormone to the same ex-
tent as that of pRNA and dRNA. The effect of hormone on the labeling of
ERNA ( Table 5 ) was strong ( 2- to 3-fold) on the guanylic, adenylic, and
uridylic residues, but limited ( 17%) on the labeling of cytidylic acid. This
Indicates different mechanisms for the two types of labeling, that of cytidylic
1-5
being due to end-group turnover, and that of the other nucleotides to de novo
chain synthesis. Since the content of CMP in tRNA is roughly equivalent :0
that of GMP, we were able to correct the data for CMP for the extent of
labeling due to chain synthesis. When this is done it becomes apparent
that there is no hormone effect on terminal nucleotide turnover, in contrast
to tho synthesis of tRNA which is affected by the hormone to the same ex-
tant as pRNA and dRNA.
The possibility that increased laheling of RNA could be due to an
hormonal stimulation of the labeiing of precursor nucleotides would appear
to be negated by the finding that end-group turnover of tRNA is not hormone
sensitive. In addition, we have isolated the AMP of liver hornogenates and
determined the radioactivity of its phosphate as a function of hormone treat-
ment. When the usual correction for variation in Pi specific activity was
made, there was no change in the specific activity of AMP, although there
was the usual Increase in that of RNA ( Table 6). We therefore are confident T-6
that the changes in RNA labeling we observe are in fact due to increased
synthesis of RNA and not to precursor perturbations.
The pattern that emerges from this study is clear in one respect,
namely, that synthesis of all the known functional types of RNA 18 stimu-
lated by adrenal hormone. Still far frora clear, however, is the relationship
of this stimulation to what appears to be selective enzyme induction. The
oft-oited model of control of genetic activity formulated by Jacob and
Monod (45) Umits control to the synthesis of mRNA, and the hormonal in-
duction studied here is clearly more complex than this moments. The model
cited was developed from studies of enzyme Induction and virus-infection
in microbial systems, and it is entirely possible that control of enzyme syn-
thesis in these instance? :: Quite different from that operative in mammallan
cells. However, in the cont of our present knowledge of protein synthe-
sis, it is difficult to understand why new synthesis of both tRNA and rRNA,
as well as mRNA, should be required in order to selectively increase the
rate of synthesis of a few enzyme proteins.
An alternative that must be seriously considered is that the selectivity
of enzyme Induction by hydrocortisone 18, in fact, only apparent. The
generalized increase in RNA synthesis that follows steroid administration
is consistent with a general increase in protein synthesis, rather than with
a selective effect on the synthesis of some proteins. Were such an overall
increase in protein synthesis to occur, differential increases in specific pro-
teins would be expected since the rate and extent of increase of any particu-
lar enzyme is then determined only by its turnover time ( rate of enzyme
degradation) and the amount of enzyme present before increased synthesis
begins. The available evidence suggests that this is, indeed, the case
in the inductions of hepatic enzymes by glucocorticoids. Rapid and extensive
increases are observed in tyrosine transaminase and in tryptophan pyrrolase,
are
and the half-lives of these enzymes in of the order of a few hours.(3,4) In
contrast, a modest Increase in glutamic-pyruvic transaminase requires
3.5
several days, and the half-Life of the enzyme is in days.(2) There is,
thon, no evidence of a differontial efíect of hydrocortisone on the rate of
synthesis of these enzymes.
stribe
mo's gettinerantem
One predictable consequence of these considerations is that any means
by which hepatic protein synthesis to stimulated should result in apparent
Inductions of the rapid turnover onzymes of the liver. Preliminary experi-
ments to test this hypothesis have revealed that it is possible to effect in-
arnases in tyrosine transaminase and tryptophan pyrrolase with agents other
than adrenal steroids. As yet the increases we have observed are small,
ao in the effect of ethanol shown in Table 7; tryptophan pyrrolase undergoes T-7
changes of the same order of magnitude as those shown for tyrosine transami-
nase. Similar effects can be observed in vitro on the Radyber hepatoma growing
in culture, so it is unlikely that residual steroids or adrenal tissue surviving
adrenalectomy can be invoked to explain these results. The mechanism of
this ethanol effect 18 unknown, but that it is prevented by actinomycin suggests
that new RNA synthesis is required.
Summary
Fractionation and characterization of the pulse-labeled RNA from livers
of rats treated with hydrocortisone demonstrates that the hormone increases
the synthesis of ribosomal and transfer RNA as well as a DNA-Uke RNA. The
hormone did not affect the rate of labeling of transfer RNA due to turnover of
the -pCpapa terminus of the polynucleotide chain. Labeling of acid-soluble
nucleotides (AMP) was similarly unaffected. The general nature of the hor-
mone effect on RNA synthesis suggests that ensyme Induction may simply re-
flect a general increase in hepatio protein synthesis. The rapidly tun ng
over enzymes tyrosine transaminase and tryptophan pyrrolase can be induced
to a limited extent by ethanol in a steroid-independent and actinomycin-sensitive
fashion.
Table 1
'P Base Composition of Pulse-Labeled Liver Nuclear KNA
Time After
Hydrocortisone (hr)
Percent of Total Radioactivity in
CMP GMP AMPUMP
A
Ratio
+ U/G + C
0.89
27
27
27
26
27
25
22
21
23
25
25
25
0.85
23
0.92
Bulk liver RNA
0.63
Liver DNA
1.34
Table 2
Fractionation of Pulse-Labeled Nuclear RNA
antation
Fraction
No.
Extraction
Conditions
Totalp
Sedi
Peak of ºp-RNA
Composition
of "P-RNA
A + U/G + C
Type of
RNA
48
0.69
33 TRNA
67 ?
-l0s, Disperse
0.72
90 PRNA
10 D-RNA
-20s, Disperse
0.93
70 PRNA
30 D-RNA
4
85° + SDS
85° + SDS
28
-308, Disperse
308
Dispers e
1.09
1.09
40 PRNA
60 D-RNA
GO DRAMA
*Modified from Georgiev, G. P., et al.es
Table 3
Hydrocortisone Effect on Pulse-Labeling of High Molecular Wought
RNA Fractions
Fraction No.
Total Radioactivity
- Hydrocortisone Hydrocortisone
Increase
55,800
1.8
102,500
185,000
133,000
91,700
64,500
2.0
2.1
Hydrocortisone treatment was for two hours.
;:
cit.
i s:**'
..si
Table 4
Fractionation of Low Molecular Weight 32p-RNA*
Fraction
Totalp
Acceptor
Activity
Sedimentation
Characteristics
mposition (%)
CMP GMP AMP UMP
M NaCl soluble
33
+
70
8
8
14
M NaCl Insoluble
48. Disperse
29
20
27
24
*Modified from Greenman, Konney, and Wicks.14)
Table 5
:
.
.
.
r
.
Hydrocortisone Effect on Pulse-Labeling of Transfer RNA
1
.
...
.
.
.
.
Time After
Hydrocortisone (hr)
Total 32p (cts/min) in
GMP AMP UMP CMP
Corrected
Terminal CMP
.
.
.
..
2400
325
948
264
685
640
1203
2725
3200
2252
*Corrected for variation in Pi specific activity.
Table 6
Hydrocortisone Effect on Labeling of RNA and Acid-Soluble AMP
Tin. After
Hydrocortisone (hr)
Specific Activity
PIAMP RNA
Relative specific Activity
AMP RNA
7.16
211.8
8680
29.6
1210
7.88
242.5
18340
30.8
2330
* Expressed as cts/min/uumole for Pi; cts/min/mmole for AMP; cts/min/mg for RNA.
The RNA was the salt-insoluble, low molecular weight fraction.
Spoolfic Activity corrected for variation in that of Pl.
Table 7
Y
?
?
Induction of Tyrosine Transaminase by Ethanol in Adrenalectomized Rats
S
Time After
Ethanol (hr)
Tyrosine Transaminase Activity (units/mg protein)
- Actinomycin + Actinomycin
.
..
-
w No o
15 (+1)
21 (44)
24 (+1)
24 (+1)
15 (+1)
14 (41)
16 (44)
11 (41)
Two ml of a 10% solution of ethanol was given intraperitoneally.
Actinomycin ( 100 ug/100 grams ) was given 1 hr prior to ethanol. The
animals were adrenalectomized and deprived of food the day prior to the
experiment. Numbers in parentheses represent the range of experimental
values obtained using 2 or 3 animals in each group.
ch
UN
:
7
VO
201
.
2
.
Legend to Figures
Figurel.
Enzyme induction and retention of hydrocortisone. Adaptad
from Kenney and Flora. (16)
.
.
.
*
.
.
Figure 2.
Rates of synthesis of nuclear and cytoplasmic RNA during
onsyme Induction. From Kenney and Kull.16)
Figure 3.
p-labeling of RNA.
Hydrocortisone effects on long term
From Kennoy and Kul. (6)
Hlyn
26
.
RY-
''
Sipas
References
ni!
1.
F. T. Kenney, Induction of tyrosine-a-ketoglutarate transaminase
in rat liver. IV Evidence for an increase in the rate of enzyme
puhta
synthesis. J. Biol. Chem. 237: 3495 (1962)
H. L. Segal and X. S. Kim, Glucocorticoid stimulation of the bio-
synthesis of glutamic-alanine transaminase. Proc. Natl. Acad.
801., U.S. 50: 912 (1963)
R. T. Schimke, E. W. Sweeney, and C. M. Berlin, An analysis
of the kinetics of rat liver tryptophan pyrrolase Induction: the
significance of both enzyme synthesis and degradation. Blochem.
Biophys. Res. Commun. 15: 214 (1964)
W. E. Knox, M. Ogata, N. Hasegawa, and X. Tokuyaraa, The
hormone and substrate types of Induction of Uver tryptophan
pyrrolase. Abstracts Vith International Congress Biochem. IX-45
(1964)
6.
M. Feigelson, P. R. Gros&, and P. Folgelson, Early effects of
cortisone on nucleic acid and protein metabolism of rat liver.
Biochim. Biophys. Acta 55: 495 (1962)
F. T. Kenney and F. J. Kull, Hydrocortisone-stimulated synthesis
of nuclear RNA in enzyme Induction. Proc. Natl. Acad. Sci., U.S.
50: 493 (1963)
0. Greengard, M. A. Smith and G. Acs, Relation of cortisono and
synthesis of ribonucleic acid to induced and developmental enzyme
formation. J. Biol. Chem. 238: 1548 (1963)
8.
10.
R. P. Perry, The role of the nucleolus in RNA metabolism and
other collular processos. Natl. Cancer Inst. Monograph 14:
73 (1964)
K. Scherrer, H. Latham, and J. E. Damell, Demonstration of an
unstable RNA and of a precursor to ribosomal RNA in Hela cells.
Proc. Natl. Acad. Sci., U.S. 49: 240 (1963)
T. Tankmoki and G. C. Mueller, Synthesis of nuclear and cyto-
plasmic RNA of Hola cells and the effect of actinomycin D.
Blochem. Blophys. Ros. Commun. 9: 451 (1962)
G. P. Georgiev, O. P. Samarina, M. 1. Lerman, M. N. Smirnov
and A. N. Severtzov, Blosynthesis of messenger and ribosomal
ribonucleic acids in the nucleolochromosomal apparatus of
animal cells. Nature 200: 1291 (1963)
M. Revel and H. H. Hlatt, The stability of liver messenger RNA.
Proc. Natl. Acad. Sci., U.S. 51: 810 (1964)
R. P. Perry, P. R. Srinivasan, and D. E. Kelley, Hybridization
of rapidly labeled nuclear ribonucleic acids. Science 145: 504 (1964)
D. L. Greenman, F. T. Konney, and W. D. Wicks, On equating
low molecular weight RNA with transfer RNA. Biochem. Biophys.
Rus. Commun. in praus
F. Jacob and J. Monod, Genetic regulatory mechanisms in the
synthesis of proteins. J. Mol. Biol. 3: 318 (1961)
F. T. Kenney and R. M. Flora, Induction of tyrosine-a-ketoglutarate
transaminase in rat liver. I Hormonal naturo. J. Biol. Chom. 236:
2699 (1961)
12.
13.
15.
16.
(cts/min/g-LIVER!
HEPATIC HYDROCORTISONE
6000
+1000
800
049'//

FIG. 1
al
TIME AFTER HYDROCORTISONE (hr)
O NON-INDUCED
RANGE
-
-.....
-
-
- -
-
-
-
0-----
---
(UNITS /mg PROTEIN)
· TYROSINE--KETOGLUTARATE TRANSAMINASE
---
-
-
€
a
-
:.
..
.
..
...
.
..
-
...
.
.
TYROSINE-Q-KETOGLUTARATE
RELATIVE SPECIFIC RADIOACTIVITY TRANSAMINASE UNITS/mg
OF RNA
PROTEIN
O. 60
TIME AFTER HYDROCORTISONE (min)
120 180 240
CYTOPLASMIC
NUCLEAR
FIG.
LB1 21


-
-i
'
.-
.-.
.
.
-
-
--

-
-
-
-
-
SNY
MAY
4
UN AV
..
.
.
25
...
4,
12,189
,189
..
NUCLEAR
CYTOPLASMIC
RELATIVE SPECIFIC RADIOACTIVITY
OF RNA


0
·
1 2 3
TIME AFTER HYDROCORTISONE AND P32 (hr)
O
FIG. 3
DATE FILMED
121/ 1 /164







.
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C
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