Download Intracellular Distribution of Radioactivity in Nucleic Acid tration of

Intracellular
Distribution
Nucleotides
of Radioactivity
and Proteins Following
in Nucleic
Simultaneous
Acid
Adminis
tration of P32and Glycine_2_C@*t
EVELYN
PEASE
TYNER,
CHARLES
HEIDELBERGER,
AND G.
A.
LEPAGE
(McArdle Memorial Laboratory, The Medical School, University of Wisconsin, MadisOn 6. Wis.)
Because
INTRODUCTION
@
the danger
of contamination
liver cell fractions.
Considerable
attention
tion
comparison
was
extended
by Jeenen
and
Szafarz
(@9),
of nucleic
acids by impurities of high specific activity has
been clearly demonstrated
and recognized (14),
For some time we have been interested in a
comparison of nucleic acid and protein synthesis in
normal and neoplastic tissues (@3, 38, 34, 49) and
in the mechanisms of these processes. To that end,
the incorporation
of glycine-@-C'4 into proteins
and chromatographically
purified nucleic acid
purines has been studied in some detail. It ap
peared desirable to extend the scope of the project
to an investigation of the simultaneous inconpora
tion of precursors of two different moieties of the
nucleotide molecule into the nucleic acids of van
ous structural and functional elements of the cell.
For this purpose,
@32and glycine-@-C'4 were
chosen.
It is now well established that nibonucleic acid
(RNA) behaves in an intracellulanly heterogeneous
fashion. In 1948 Marshak (36) reported that con
siderably more @32
was incorporated into the RNA
of the nucleus than of the cytoplasm. This observa
some
workers
have
chosen
chromatographically
Volkin
to
purified
and co-workers
work
only
compounds.
(48, 51) have
with
Thus,
determined
the specific activities of mononucleotides,
isolated
from DNA and RNA, after the administration
of
@32or
of
formate-C'4.
They
found
that
mononu
cleotides from the same nucleic acid differed
among themselves in specific activity. Similar re
sults were obtained in a time study by Boulangen
et at. (7). Hultin et a!. (@6)have shown small dif
ferences among the nucleotides. Davidson, Mc
Indoe, and Smellie (15) have combined the tech
nics of cell fractionation
with those for the isola
tion of individual nucleotides in a preliminary ne
port on the uptake
of the
of
@32
by ribonucleotides
has been
incorporation
given
(sometimes
in rat
to a
in
correctly referred to as “turnover―)of precursors
who obtained three cytoplasmic fractions by dif
ferential centnifugation
and found that the
@32into DNA and RNA of nongrowing tissue. Brues
et al. (10) showed a fivefold
greater specific ac
specific activity of the RNA differed in each frac
tivity of RNA as compared to DNA following ad
tion. Such a difference was not observed by Rei
ministration of p32; Hammarsten and Hevesy (@)
chard (4@), using glycine-N'5 as a precursor. An cx
report factors of 10—38for similar cases. Brown
tensive study of @32
specffic activity-time relation
and co-workers (9), using labeled adenine, ne
ships in cell fractions
of desoxynibonucleic
acid
(DNA), RNA, phosphoproteins, and inorganic ported an incorporation ratio for liven RNA :DNA
phosphate has been carried out by Barnum and
of 100 : I . In striking contrast to those results, we
Huseby (3) and Barnum et @1.
(4), which has en
have reported (@3, 33, 49) that glycine-@-C'4 is in
abled them to perform calculations which bean on corponated
rapidly and extensively
into the pu
rines of both nucleic
acids with a specific
activity
turnover rates of the processes involved. This
work further established the intracellular hetero
ratio RNA : DNA of about
: 1. Independent
stud
geneity of nucleic acids.
ies by Elwyn
and Spninson
(19) with glycine
and
formate, Abrams (@)with the same precursors, and
S This work was supported
by a grant
from the Wisconsin
Totter et al. (48) with formate supported this find
Section of the American Cancer Society, and by the Alexander
ing of appre@ciable synthesis of DNA punines. In an
and Margaret Stewart Trust Fund.
t An abstract of part of this work has been published in attempt to reconcile these differences we have
Fed. Proc.,11:800,1952.
Received for publication November 5, 1952.
suggested
mechanisms
(@3, 49)
that
of DNA
two
separate
synthesis
must
pathways
or
exist. One
186
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TYNER
et al.—Radioactivity
in
could involve the synthesis of nucleic acids from
preformed
adenine,
canbohydrate,
and phos
phonus; the other, the incorporation
of small
molecule building blocks into a skeleton already
containing the carbohydrate
and phosphorus moi
eties. Support for this concept appeared to have
been provided by the experiment of Funst and
Brown (@0), who administered
glycine-N'5 and
adenine-8-C'4
simultaneously
and found con
siderably more N'5 than C'@ in the DNA. The
present work appears to show that this concept is
incorrect.
In order to clarify these and other points, the
present comprehensive
investigation
was under
taken. Glycine-@-C'4 and
@32
were administered
simultaneously to normal and tumor-bearing rats.
The livers and tumors were subjected to cell frac
tionation. Mononucleotides
were isolated from the
DNA and from the RNA of each cell fraction, and
their specific activities with respect to each isotope
were determined. Several time intervals were em
ployed to increase the validity and scope of this
work. In addition, the specific activities of the
ultimate precursors (glycine and inorganic phos
phonus) were determined as well as those of the
acid-soluble and -insoluble phosphorus. The C'4
contents of the proteins of each cell fraction were
determined. The tumor chosen for study was the
Flexner-Jobling
carcinoma,
a rapidly
growing,
highly anaplastic rat tumor. It was hoped that by
this approach a considerable amount of new in
formation might be gained concerning the bio
chemical mechanism of nucleic acid synthesis and
the differences between tumor and liver. This in
formation might to some extent bridge the gap
between the biochemical and cytochemical
ap
preaches. Some of these hopes have, at least in
part, been realized.
EXPERIMENTAL
Female rats,' weighing 150—170gm., were used.
Half of these had 10-day-old multiple, subcutane
ous transplants of Flexnen-,Jobling carcinoma, and
the other half were nontumon-beaning controls. A
single intrapenitoneal
injection of 33.4 @c.of @32
2
and 13.3 [email protected] glycine-@-C'4 (0.8 mg.)3/100 gm
body weight was given to each rat; three animals
were used in each experiment. In both the normal
and tumor-bearing
series, the animals were sacri
1 Obtained
from
the
Holtzman-Rolfsmeyer
Rat
Co.,
Madi
son,Wis.
I The
P@'
Department
was
supplied
of Medicine,
to
us
by
University
Dr.
E.
C.
Aibright
of Wisconsin,
of
the
on alloca
tion from the U.S. Atomic Energy Commission.
I The glycine-2-C'4 was purchased from Tracerlab, Inc.,
on allocation from the U.S. AtomicEnergy Commission.
Nucleic
AcüLi and
187
Proteins
ficed @,5, 1@,and 48 hours after injection. Stock
grain diet and tap water were provided. After the
appropriate
interval the rats were decapitated,
and their livers were perfused with cold isotonic
sucrose (0.@S M). The livers on tumors were then
pooled,
minced,
homogenized
in cold isotonic
su
crose, and carried through a modification of the
differential centnifugation procedure of Schneider
and Hogeboom (44) at 0—4°
C. The outline of the
experiment is shown in Chart 1.
Preparation of cell fractions.—The homogenate
fuged at 600—700g for 10 minutes, the supernatant
was centri
liquid was
removed,and the nuclei were resuspendedin the sucrosesolu
tion and recentrifuged twice. The nuclei were then suspended
in 2 per cent citric acid (16) and were allowed to stand at 0°for
several hours to facilitate the removal of cytoplasmic con
tamination. The combined supernatant fluids (S1) were centri
fuged at 5,000 g for 10 minutes to obtain mitochondria.
The
fraction was resuspended and centrifuged at 7,000 g and again
at 9,000 g. Under these conditions the “poorly
sedimented
layer―(40) was included in the mitochondrial fraction, since it
has been shown (31) that this material contains a larger pro
portion of RNA than do the larger mitochondria.
The mito
chondria were then suspended in water. The combined super
natant liquids (S2) were sedimented in an air-driven centrifuge
at 120,000 g for 36 minutes. The supernatant liquid (S,) was
removed, and the microsomes were resuspended in water. The
nuclei, which had been left in citric acid, were centrifuged,
washed again with citric acid, and then washed several times
with 2 per cent acetic acid untilthe supernatant fluid was clear
and the nuclei
appeared
free of cytoplasmic
contamination
when observed under the phase microscope. These washes
were pooled; analyses (43) showedlittle DNA, but considerable
RNA and protein present. Analyses were routinely carried out
on all fractions and are summarized in Table 1. “Tumor-bear
ing liver―refers to the livers of tumor-bearing rats.
An aliquot of the original homogenate was used for the de
termination
of thespecific
activities of the acid-soluble and
acid-insoluble phosphorus and of the free glycine (28).
Isolation of nucleifroin
liver using a nonaquseus
medium.—
In order to investigate the changes in specific activities of the
protein and RNA that are extracted during the preparation of
the citric acid-washed nuclei, it appeared desirable to isolate
nuclei with a nonaqueous medium. Our application of the modi
fication of Dounce ei al. (19) of Behrens' technic (5) led to
“nuclear―
preparations containing distorted nuclei that were
grossly contaminated by cytoplasmic material. The ethylene
glycol procedure of Neff (37) was found to yield well formed
nuclei, admixed with cytoplasmic fragments that could not be
removed by differential centrifugation or by filtration through
bolting silk and flannelette. However, some data were collected
with this preparation and were compared to “citric
acid nuclei―
obtained from the same liver mince.
Isolation of nudeic acids and proteins from cdl fractions.—
The preparation of nucleic acids was carried out by a modifica
tion of the method of Hurlbert and Potter (27) as follows:
Each cellfraction at 0°was made 0.4 N with respect to perchlo
nc acid, and the precipitate was centrifuged and washed twice
with 0.4 N perchioric acid and then twice with 95 per cent
ethanol. Lipids were extracted at 40―—50@
by three washes with
8: 1 ethanol-ether. The residue was suspended in 10 per cent
NaCland neutralized with NaOH; a smallamount of saturated
NaHCO3 solution was added to maintain a slight alkalinity.
The supernatant liquid after centrifugation was discarded, the
residue
was resuspended
in 5 volumes
of 10 per cent NaCl
(neutral or slightly alkaline), and the mixture was heated for 1
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1953 American Association for Cancer Research.
188
Cancer Research
hour at 1000to extract the nucleic acids. After centrifugation,
the supernatant liquid was filtered through glass wool, and the
process was repeated on the residue with 3 volumes of the
NaCl solution and with 30-minute heating. The residue was
washed twice with 5 per cent trichloroacetic acid and twice
with acetone, and was dried in vacuo over sulfuric acid. This
tration of formic acid from 0—5M (11). The four nucleotides
came off the column in the order cytidylic, adenylic, guanylic,
and uridylic acid, and were located by spectrophotometric
analysis of the individual tubes. Considerable information on
the purity of the compounds was gained by measuring the
ratio of the absorption
at 260 and 290 m@. Volkin it at. (52)
material was used for the determination of the C'@specific have developed chromatographic procedures for the separation
activity of the protein. The NaCl extract was treated with S
volumes of cold ethanol and allowed to stand at 0°overnight.
of the adenylic and cytidylic desoxyribonucleotides from the
corresponding ribonucleotides. When this method was applied
The precipitated sodium nucleates were obtained by centrif
iigation.
to typical radioactive desoxynucleotides,
Preparation
and analysis
of mononncleotides.—In
order to
location on the chromatogram.
Pa,
This established
that the DNA
C―
(ADENYLIC
-4 ADENINE
- ____ -@<
IGUANYLIC
DNA
CYTIDYLIC
@
the specific activities
were unchanged, and no peak was found in the ribonucleotide
+ H,PO4
—‘
GUANINE
THYMIDYLIC
@NUCLEI
-5
____
RNA
(ADENYLIC
1GUANYLIC
@‘1CYTmYLIC
(URIDYLIC
-4
ADENINE
a
—4 GUANINE
t@
PROTEIN
@1
RNA
LIVER
OR TUMOR
____
(ADENYLIC
—5 ADENINE
J GUANYLIC
-5
GUANINE
-5
ADENINE
a
a
I CYTIDYLIC
(URIDYLIC
MITOCHONDRIA--@
t@
PROTEIN
(ADENYLIC
HOMOGENATE
11RNA
@
MICROSOMES
—‘
____
a
a
—@
GUANINE
I GUANYLIC
-)@
CYTIDYLIC
URIDYLIC
I PROTEIN
(ADENYLIC
a
—,
ADENINE
, J GUANYLIC
a
—5 GUANINE
I RNA
SUPERNATE
@
—@
CYTIDYLIC
(@LTRIDYLIC
IPROTEIN
CHART 1.—Isolation of proteins, nucleic acid nucleotides and purines from cell fractions
hydrolyze
the RNA samples to mononucleotides
and to accom
nucleotides were contaminated with less than 0.05 per cent of
plish the separation of DNA in the nuclear fraction from RNA,
@
RNA nucleotides.
After the spectrophotometric analyses were carried out,
each sodium nucleate sample was treated with 0.1 N NaOH
(approximately 1 ml/10 mg of sodium nucleate) at 37°for 15— tubes were pooled so that duplicate samples of each nucleotide
were obtained. These were dried in vacuo over CaCl2 and
20 hours. (it was found necessary
to reduce the time of this
NaOH under an infrared lamp to remove the formic acid. (Heat
incubation to 10 hours for the preparations
from tumor.) The
was not used on the DNA preparations.) Each dried nucleotide
samples were cooled to 0°,and concentrated HC1 was added to
sample was dissolved in 5.0 ml. of 0.1 N HC1, and was counted
an excess concentration of 0.1 as. The precipitated DNA of the
for P'@in an annulus counter.
This counter had an efficiency
nuclear fractions was centrifuged rapidly, and the supernatant
of 17 per cent for P@, and the glass walls were of sufficient
fluid (containing
nuclear RNA) was removed and filtered.
The DNA was washed carefully with cold dilute HC1 and dis
solved in 0.1 N NaOH. Analysis (48) showed that there was
consistently less than 1 per cent RNA contamination in the
DNA preparations. The RNA solutions after neutralization
were ready for ion-exchange chromatography. The DNA was
subjected to a double enzymatic hydrolysis with DNA-ase and
phosphatase (28, 35, 52) to produce the desoxymononucleo
tides, which were then separated
raphy.
by ion-exchange
chromatog
The chromatographic procedure was a modification of the
methods of Cohn (13) and involved the use of a Dowex-1-for
mate column and elution with a gradually increasing concen
thickness to absorb allthe C―,so that only the PMwas counted.
After appropriate dilution, the concentration of nucleotide in
each solution was determined spectrophotometrically with the
use of the constants of Hotchkiss (24), and the specific activity
of the @M
in pc/pM was calculated.
4 Obtained
from
Radiation
Counter
Laboratories,
Skokie,
Ill., Mark 1, Model 70. The side arms were modified so that the
counter contained a constant volume of liquid.
a We acknowledge the assistance of Mr. Sherwyn Woods
for the @U
counting, and of Mrs. Edith Wallestad, Miss Nancy
Lake, and Mrs. Dorothy McManus for the C―determinations.
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1953 American Association for Cancer Research.
TYNER
et al.—Radioaciivity
in Nucleic
Preparation and analysis of the purines.—The purine nu
cleotides were hydrolyzed in 1 N HC1 for 1 hour (RNA) or 20
minutes (DNA). The samples were then diluted 1 :4 with water
and a lox molar excess of ;ca@rierphosphoric acid was added.
The mixtures were put on small (6 X 10 mm.) columns of
Dowex-SO resin. The columns were washed with 25 ml. of
water and 1.5 ml. of 2 N HC1, which removed allthe phosphate.
The purines were then eluted with 4 ml. of 6 NHC1, evaporated
to dryness in vacuo,dissolved in 0.05 N HCI, plated by evapora
tion onto tared aluminum discs, and counted for C―.No P@
was found to contaminate these preparations. The purines
were eluted from the aluminum disc with 0.1 N HC1 and de
termined spectrophotometrically
(49). The specific activities
in sc/pM were calculated. These measurements of radioactivity
were made in internal flow counters and were corrected for
self-absorption.
They were counted sufficiently long to reduce
189
A@kL@and Proteins
in contrast to the results,
nucleic acid analyses.
just
cited,
for the
Calculation and gra@phie
representation of results.
—In the case of the punines on nucleotides,
specific
activities
were first
of compound.
absolute
These
values
obtained
were then
the
in cpm/zM
converted
by the application
into
of the known
counter efficiencies. The C―specific activities were
then multiplied by @.50,which corrects the values
TABLE 1
NUCLEIC
ACID CONTENT
OF TISSUES
MG/GM TISSUE (WET WEIGHT)
the statistical error to less than 10 per cent.'
“Tumor
Measurenwnt of the specific activity of the proteins.—The
finely ground protein samples were suspended in 25 per cent
ethanol, spread evenly on aluminum discs, and dried in vacuo.
They adhered firmly to the plates. They were counted with an
end-window counter with and without aluminum absorbers of
bearing―
liver'
Normal liver
Tumor
DNANuclei1,3501,610RNANuclei240424Mitochondria7821,050Microsomes1,6852,215Sup
3,690
1,220
292
various thicknesses to differentiate the C―and P―.By means
of empirical factors, the specific activity
the protein was calculated.
(pc/mg)
of the C―in
910
770
* Livers
from
tumor-bearing
rats.
RESULTS
Analyses of the biological material.—To define
rigorously
the
nature
of the
biological
TABLE 2
material
with which we were dealing, analyses for nucleic
acids and proteins within the various cell fractions
were carried out routinely throughout the series of
experiments. The results are shown in Tables 1 and
@2,
which represent the average of values obtained
from at least twelve animals. Data similar to these
have been reported many times from a number of
laboratories, but the discussion will be restricted
to the analyses at hand. It will be noted from
Table 1 that there is a markedly larger quantity of
PROTEIN
CONTENT
OF TISSUES
IN
MG DRY WT/GM TISSUE
(wi@T WEIGHT)
“Tumor
bearing―
liver
141.0
6.7
26.2
Normal liver
173.0
5.2
23.1
Homogenate
Nuclei (citric acid)
Nuclear citric acid
washings
Mitochondria
36.6
21.1
31.1
Microsomes
Supernatant
Tumor
109.0
18.5
12.5
4.9
35.5
23.9
30.2
10.5
27.3
DNA and nuclear RNA pen gram of tissue in the
@
tumor as compared to the normal liver and that
“tumor-bearing―liver is intermediate.
There is
to a dose equivalent
considerably
1O3/@iM,
less mitochondnial
and
microsomal
RNA in the tumor and more in “tumor-bearing―
liver, as compared to normal liver. With regard to
the supernatant
fraction, the tumor has a higher
content of RNA than either the normal or “tumor
bearing― livers.
In Table
are shown the protein contents of the
tissues, and once again the quantity in the mito
chondnia of the tumor is strikingly smaller. It is
also
seen
that
the
majority
of the
protein
in the
nuclear fractions from liven is extracted during the
citric acid washes. Although it was not feasible to
measure the absolute protein content of nuclei
prepared by the ethylene glycol procedure, the
ratio of protein to DNA was approximately twice
that found for the citric acid-washed nuclei. The
protein contents of the fractions from normal and
“tumor-bearing―livers did not differ significantly,
p32• All
specific
in radioactivity
activities
a quantity
that
are
to that
expressed
of the
in
is comparable
to
@c X
specific
activities computed as the per cent of the ad
ministered dose. The specific activities are graphed
as a logarithmic
plot
against
time.
Since
measure
ments were made at only four time intervals,
points
are connected
tempt
was
made
by straight
to construct
the
lines, and no at
smooth
curves.
To calculate the total radioactivity per gram of
tissue, it was necessary to know the nucleotide
composition of the nucleic acids. The values used
were those of Wyatt (53) for DNA and of Volkin
and
Carter
(50)
for
RNA.
Although
the
ion-ex
change separation of the nucleotides in these cx
peniments was designed from the point of view of
purity, rather than that of quantitative
recovery,
it appeared
that
in the tissues
and
fractions
we
have studied the nucleotide composition is very
close to that given in Table 3. Knowing the spe
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Cancer Research
190
cific activity, the nucleotide composition (Table
3), and the nucleic acid content (Table 1), the
total radioactivity,
expressed as @&c
X 10@/gm of
tissue was calculated. These data are also plotted
logarithmically
against time.
Acid-soluble and -insoluble phosphorus and un
combined glydne.—The specific activities of the
acid-soluble
and -insoluble phosphorus
and of free
TABLE S
the “true―
pooi size with any degree of precision,
but it is felt that, on the basis of the presently
available data, the conclusion that the phosphorus
and glycine pool sizes are similar is justifiable.
This result makes the interpretation
of subsequent
data more feasible.
Chart S shows the total radioactivity
of the
acid-soluble and -insoluble phosphorus. Again, the
two types of livers gave similar results. The total
@32in
PER CENT Coan'osrrloN
oi@NUCLEOTIDES
IN NUCLEIC ACIDs
the
acid-insoluble
fraction,
which
includes
nucleic acids and phosphoproteins,
increases with
time, although the acid-soluble P and the sum of
the two decreases.
(Wyatt [531,Volkin and Carter [50])
DNA
RNA
Adenylic acid
Guanylic acid
Cytidylic acid
Uridylic acid
28.7
17.9
21.4
32.8
20.4
55.9
Thymidylic acid
Methylcytidylic
acid
28.4
1.1
Adenylic
acid
@32
fr@
nucleic
acids.—The
spe
cific activities of adenylic acid-P32 from the indi
vidual cell fractions are shown in Charts 4 and 5.
It is evident that the specific activities of all frac
tions of the tumor are higher than the correspond
15.3
TIME-HOURS
CHART 2
glycine in “tumor-bearing―liver and in tumor are
shown in Chart 2. There is no appreciable differ
ence between the livers from normal and tumor
bearing rats with respect to these substances. The
specific activities
for tumor
are significantly
higher
than those for the livers, which shows that the
tissues are not in complete equilibrium with re
spect
@
to the
ultimate
precursors,
even
after
48
hours.
Because the specific activity-time
curves of the
acid-soluble
phosphorus
and free glycine show
good agreement at almost every time studied, and
because
the
doses
of the
labeled
precursors
have
been equated, we conclude that the pool sizes of
these precursors must be similar. In this type of
experiment
it is difficult
or impossible
to measure
ing samples from liver and that there is less spread
among the fractions in tumor than in liven. This is
true for all four nucleotides. It will also be noted
that there is no significant diff@rence between the
RNA of normal and “tumor-bearing―liven in any
cell fraction.
In the case of the DNA adenylic acid-P32 it is
found that, for the tumor, there is a considerable
increase of specific activity with time and that it
corresponds
fairly
closely
with
the
cytoplasmic
RNA. On the other hand, in normal liver, the
specific activity of the DNA at
hours appears
surprisingly high and increases very little more
with time, so that at 1% and 48 hours it is much
lower than the cytoplasmic RNA. It is felt that the
data at all times are valid, because it has in
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1953 American Association for Cancer Research.
TYNER
@
et al.—Radioactivity
in
variably been shown that there was less than 1 per
cent RNA contamination
in these samples and, in
certain cases, less than 0.1 pen cent.
Examination
of the curves for the RNA ade
nylic-P32 shows, in agreement with the work of
others (3, 4, 15, @9),that the nuclear fraction has
a much higher specific activity than the cyto
plasmic fractions; at
hours it is 45 times higher
than the specific activity of the mitochondrial
adenylic acid. In the liver, the specific activity of
the nuclear RNA decreases with time, whereas in
the tumor it increases. At early times, the order of
decreasing specific activity always found was nu
Nucleic
Aeids
and
191
Proteins
clear, supernatant, microsomal, and mitochondnial
RNA. That this is true for all the nucleotides is
illustrated
by
Chart
6, which
shows
cytidylic
acid-P32, although there are individual quantita
tive variations among the individual nucleotides.
By means of these chromatographic
technics we
are able to find significant differences in specific
activity
between
the
mitochondnial
and
micro
somal fractions, which others have been unable to
observe (8, 4, 4@).
In Chart 7 is shown the total adenylic acid-P32
per
gram
of tissue
versus
time.
In the
normal
there is a decrease with time of the total
liver
@32
in the
TOTAL RADIOACTIVITY P@2 OF ACID-SOLUBLE B ACID-INSOLUBLE PHOSPHORUS
U%J%I%d
500
@
;jj
SUM
@
:@-..-.-.-__c@
@
@
ACtD-SOLUBL@@
@
@
100
.0--
0--
D@
‘0
@o
--0
,,-ACID-INSOLUBLE
ACID-INSOLUBLE
0'
@‘
50
NORMAL LIVER
TUMOR
(
TIME - HOURS
CHARTS
I
CHART 4.—N=nucleus;
S=supernatant
fluid; M=mitochondria;
m=microsomes
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1953 American Association for Cancer Research.
19@
Cancer Research
nuclear RNA fraction, whereas there is an in
crease in the cytoplasmic fractions. The relative
position of the cell fraction with regard to total
radioactivity
is different from that found in the
specific activity curves; this is due to the differ
ences in nucleic acid content of the various cell
fractions.
There is a considerable difference between the
total
@32
in the adenylic acid of the liver and
tumor. In liver, a nongrowing tissue, the total
isotope incorporated
into the DNA is relatively
small compared to the RNA. However, in the
rapidly growing tumor the total
@32
in the DNA
exceeds
the
sum
of the
activities
in all the
RNA
fractions at 1@ and 48 hours. This observation,
which is consistent for all four nucleotides and
both purines, reflects the increased DNA content
of tumor tissue as well as the net synthesis of DNA
that must be required for cell division.
Adenine and guanine-C'4 from nucleic acids.—
SPECIFIC ACTIVITY OF ADENYLIC-P@2FROM RNA OF CELL FRACTIONS
CHARTS
RNA
8 DNA
OF CELL
FRACTIONS
CHART 6
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1953 American Association for Cancer Research.
TOTAL RADIOACTIVITY OF ADENYLIC-P32 FROM RNA 8 DNA OF CELL FRACTIONS
TIME- HOURS
CHART 7
SPECIFIC ACTIVITY OF ADENINE-C14 FROM RNA & DNA OF CELL FRACTIONS
CHART
8
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1953 American Association for Cancer Research.
Cancer Research
194
. The
specific
activity-time
curves
for
scure until further
adenine-C'4
derived from the nucleic acids (Chart 8) bear a
strong resemblance to those of adenylic acid-P32,
indicating
at least
a similarity
in the
behavior
of
the punine and phosphorus moieties of the nucleic
acid molecule. The tumor is again consistently
higher
than
activity
liven and the decreasing
among the cell fractions
supernatant,
microsomal,
RNA
specific
is also nuclear,
and mitochondnial.
The
experimentation
is carried out.
Specific activity of the proteins.—The specific ac
tivities (C'4) of the proteins of cell fractions are
shown in Chart 10. These protein samples have
not been further separated or characterized,
and,
hence, are defined only by the method of isolation.
The specific activities of the tumor proteins are
somewhat higher than those of liver, but the dif
ference is not so marked as that found for the flu
TOTAL RADIOACTIVITY OF GUANINE-&4 FROM RNA B DNA OF CELL FRACTIONS
0
C)
TIME- HOURS
CHART
@
@
9
DNA of tumor is higher than the DNA of liven. In
agreement with our earlier findings (@3, 33, 49),
the specific activity of the DNA adenine in liver is
of the same order of magnitude as the average of
the RNA adenine specific activities. This is also
true of guanine.
The total radioactivity
of the nucleic acid
guanine, as shown in Chart 9, bears a similar rein
tion to time as does the adenylic acid-P32. We find
a similar sequence among the cell fractions, and
the same accumulation of C―in the DNA of tumor
cleic acids. This is in agreement with our earlier
results (49). In sharp contrast to the situation with
the nucleic acids, where the nuclear fraction of
RNA has a much higher specific activity than the
cytoplasmic at hours, the highest specific activity
in the proteins at that time is found in the micro
somal fraction.
as was found
acid washes,
with the other
tracer.
The high total
radioactivity in the DNA guanine at hours in the
liver is difficult to explain, but, as previously
pointed out, it cannot be ascribed to contamina
tion. The significance of this fact must remain ob
Comparison betweencitrz@,
acid and ethylene glycol
nuclei.—It was of interest to determine whether
the specific activities of the protein and RNA,
known to be extracted from nuclei by the citric
are the same or different
of the residual protein and RNA.
comparison was made between
ethylene glycol nuclei, prepared
pooled livers. Table 4 shows that
from
those
To this end, a
citric acid and
from the same
the specific ac
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1953 American Association for Cancer Research.
TYNER
et al.—Radioactivity
in
tivity of the proteins was the same. Thus, the
extracted and nonextnacted protein samples are
homogeneous
with
respect
to
specific
activity.
The data for nuclear RNA appear to indicate a
heterogeneity,
since the specific activity of the
ethylene glycol nuclei is considerably
reduced.
However, it was impossible to remove all cyto
plasmic contamination
from these nuclei. The ef
fect of cytoplasm would be to reduce the specific
activity
of nuclear
RNA,
so that
any conclusion
Nucleie
Acids
@
195
Proteins
also consistent with the observations of Reddy and
Cenecedo (41) of the increased content of DNA
and mitotic figures in the livers of tumor-bearing
animals. The specific activities of adenine-C'4 and
guanine-C'4
also differ
somewhat
but
follow
the
same trend.
The primary purpose of setting up an expeni
ment of this sort was to be able to make a direct
comparison of the incorporation of two precursors
into different pants of the same molecule. Such a
comparison
SPECIFIC ACTIVITY OF
PROTEIN-C14 FROM CELL FRACTIONS
and
will only
be really
valid
when
both
precursors are administered simultaneously to the
animal, and the content of both isotopes is mean
ured in the same molecule. This comparison is
shown in Chart 1@,where the specific activities of
j
TABLE 4
\\@m
1.0
TUMOR
CoMPARIsoN OF CITRIc ACID AND
ETHYLENE
GLYCOL NUCLEI
(S-hour normal liver)
Sractric ACTIVITY
5
Citric acid
Ethylene glycol
C14: @@cX
1O'/mg
@
Protein
N
0
M@
RNA
\“@b
TUMOR BEARING LIVER@‘
‘0
Adenylic acid
Guanylic acid
S.72
2.56
Cytidylic
2.28
2.80
0.827
0.712
0. 798
0.740
NORMAL LIVER
adenylic
@
—-
So.....
@
1.0
_f
@-
-
O\
0
0@—.
IVI
.
-—-—.—-—.@@
.i.@_
.
I@
ui@@4b
—
TIME-HOURS
CHART
10
DNA:
comparison
between
@32
and C@4.—Acom
panison of the specific activity of @32
among the
various nucleotides in normal and “tumor-bear
ing―liver is given in Chart 11. There is some in
dividual variation,
but with the exception of
cytidylic acid in normal liver, which is consistently
lower, the nucleotides agree rather well. The sig
nificance
of the variations
actually
found
will be
discussed below. It will be noted that in “tumor
bearing― liter, although the specific activity of the
@32in
the
DNA
nucleotides
is
lower
at
hours
than in normal liver, at 1@and 48 hours it is mark
edly higher. This is a clear demonstration
of a
acid-P32,
adenine-C'4,
guanylic
acid-P32,
and guanine-C'4 of DNA are plotted against time.
It will be seen that there is a very close correspond
ence between the p32.. and C―-containing moieties
of the DNA nucleotide molecules in tumor, normal
liver, and “tumor-bearing―liver. This similarity is
found too consistently to be fortuitous and sug
gests
about the state of the extracted nuclear RNA will
have to await a more definitive experiment.
@
acid
Uridylic acid
C)
@
1.56
1.62
P@:pcX1O'/@sie
very
strongly
that
the
glycine
and
phos
phorus are incorporated into a DNA precursor at
the same time. This concept is strongly supported
by the data on total activities, as shown in Chart
13. Here again there is a very close correspondence
between the @32
and C―total nadioactivities within
the nucleotide molecules.
RNA: comparison between @32
and C'4.—Whena
similar comparison
is made between the two iso
topes in the RNA molecule,
a different
result is
obtained.
Since a comparison
of all RNA fractions
on the same graph would be unwieldy, considena
tion was restricted to the nuclear and microsomal
fractions. Chart 14 shows the results with ade
nylic acid-P32 and adenine-C'4, and Chart 15 com
pares
guanylic
acid-P32
and
guanine-C'4.
The
data
body and is in agreement
with the results of Payne
obtained from these two graphs are closely similar.
In tumor, we find a close correspondence between
the two isotopes in the RNA nucleotides that is
et al. (89) on uptake of
@32
in mice. This finding is
reminiscent
systemic
effect
of a tumor
on other
tissues
of the
of the situation
found
for DNA
in all
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1953 American Association for Cancer Research.
Cancer Research
196
tissues.
@
On the other
hand,
in normal
liver
there
is
a great difference between the specific activities of
the @32
and the C'4. At
hours the adenylic acid
and
guanylic
proximately
acid-P82
have
specific
guanine-C'4.
This result indicates
is incorporated
into the RNA
earlier time than the glycine.
The total RNA nucleotide
compounds
activities
@0times those of the adenine
and
fractions
ap
and
that phosphorus
precursor
at an
are similar and about twice those of adenylic and
unidylic acids, which are also approximately equiv
alent. When the total
@32
of the cell fractions of
each pair is compared, there is an astonishingly
close correspondence in every case, and the total
@32in
that
the
consistent
@32
of the individual
of tumor
is shown
in
Chart 16. It will be noted from Table 3 that in
RNA the amounts of cytidylic and guanylic acids
cytidylic-guanylic
of the adenylic-unidylic
with
the
pair
is
about
twice
pair. This result is
hypothesis
that
in tumor,
where there must be a net synthesis of RNA, the
nucleic acid molecule is rapidly assembled in lob
from preformed nucleotides, since the total isotope
found in the nucleic acid molecule corresponds so
SPECIFIC ACTIVITY OF DNA NUCLEOTIDE- P32
CHART 11
@
SPECIFIC ACTIVITIES OF DNA PURINES
PURINE NUCLEOTIDES
@—.-s ADENYLIC-P32
x
@@4DENINE-C@4
o—o
GUANYLIC-P32
@---@@oGUANINE-C14
0
C)
TI ME-HOURS
CHART
12
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1953 American Association for Cancer Research.
TYNER
exactly
et al.—Radioactivity
with the nucleotide
composition.
Thus,
in
the
rates of synthesis parallel the requirements
for
individual nucleotides by the RNA.
In the case of normal liven (Chart 17), a similar
comparison demonstrates
a correlation of total
@32and
nucleotide
correspondence
in the
composition,
but
not
the
exact
found in tumor. This suggests that
nongrowing
liver
there
may
actually
be
Nucleic
some
net
Acids
and
synthesis
197
Proteins
of RNA
molecules,
which
is
overshadowed by the turnover of the macromole
cule, as well as that of the various precursors.
DISCUSSION
One of the primary objects in this research has
been to study the biochemical mechanisms of nu
cleic acid biosynthesis,
and therefore the main
TOTAL RADIOACTIVITIES OF DNA PURINES & PURINE NUCLEOTIDES
IC
5
@I.O
“—‘CADENYLIC-P32
0——--OADENINE-C14
I')
‘0
;@o.s
C)
0.I
CHART 13
SPECIFIC ACTIVITY OF ADENYLIC@P32&ADENINE@CI4FROM RNA OF CELL FRACTIONS
0
x
0
CHART14
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1953 American Association for Cancer Research.
198
Cancer Research
effort has been devoted to a study of the sequence
of events that occur at relatively short times after
ficient information
activity-time
curves
administration
of the labeled precursors.
Although
it would be highly desirable to calculate renewal or
turnover
times, we do not feel that we have suf
Nevertheless,
it is possible, on the basis of the data
presented,
mechanism
to draw
on the shape of the specific
to justify
certain
such
calculations.
conclusions
about
the
of nucleic acid biosynthesis.
SPECIFIC ACTIVITY OF GUANYLIC-P328 GUANINE-C14 FROM RNA OF CELL FRACTIONS
CHART15
CHART
16
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1953 American Association for Cancer Research.
TYNER
et al.—Radwactivtty
in
In an examination of the data for the RNA nu
cleotides in the various cell fractions, it is apparent
that the nuclear fraction has a much higher specific
activity
@
with respect
to both isotopes
(particularly
at
hours) than do the cytoplasmic fractions.
Similar results have been found before for various
precursors
This
(3, 4, 7, 15, 18,
type
Szafanz (@9) to propose that
precursor
of the cytoplasmic
the other hand, Barnum
Acids
has led Jeener
and
the nuclear RNA
RNA fractions.
is a
On
et al. (4) have made cx
and
199
Proteins
This is in agreement with the in civo observations
of Borsook et al. (6) with labeled glycine, histidine,
leucine, and lysine, of Hultin (@5) with glycine,
and of Keller (80) with leucine. Siekevitz (47) , in
studies
@6, @7, @9,86, 88, 89, 4@).
of observation
Nucleic
with
homogenates,
has
demonstrated
that
the incorporation of labeled alanine into proteins
occurs in the microsomal fraction in the absence of
nuclei, provided that mitochondnia are added to
supply
a source of energy.
Thus,
the findings
under
these conditions are not in accord with the ideas of
Caspersson (1@), who has stated that the synthesis
TOTAL RADIOACTIVITYOF RNA NUCLEOTIDE-P32
C)
NUCLEAR ADENYLIC
@—+NUC@AR
URIDYLIC
x—@ NUCLEAR CYTIDYLIC
s—+NUCLEAR GUANYLIC
a------oSUPERNATANT CYTIDYLIC
.-----.SUPERNATANT GUANYLIC
o-.-—o MIGROSOMAL CYTIDYLIC
@-•°5UPERNATANT
ADENYLIC
.----‘SUPERNATANT
URIDYLIC
o—--oMICROSOMAL ADENYLIC
..-—.
MICROSOMAL
£
.—--.
URIDYLIC
@—---@MITOCHONDRIAL
ADENYLIC
fr—-—@MITOCHONDRIAL
MICROSOMAL
GUANYLIC
a—---ôMITOCHONDRIAL
k-@ ---aMITOCHONDRIAL
TIME -HOURS
CHART
tensive calculations which lead them to conclude
that the nuclear RNA is not a precursor of cyto
plasmic
@
@
RNA,
but
rather
that
they
both
have
a
common precursor. Our experiments are super
ficially consistent with both these alternatives, but
do not provide a means to decide between them.
It should be pointed out, therefore, that in a tenta
tive scheme of nucleic acid biosynthesis proposed
below, no implications are made as to the intnacel
lular locus or loci of the process. It is inescapable,
however, that at early times the RNA of the nu
cleus has a much higher specific activity, with re
spect to both building blocks here investigated,
than do the particulate or soluble RNA from cyto
plasm.
In sharp contrast to the situation just cited for
the nucleic acids, a different intracellular distnibu
tion of protein specific activities is observed. At
hours the specific activity of the microsomal pro
teins is higher than that of the other cell fractions.
17
of proteins takes place in the nucleus, most prob
ably in the nucleolus, with the proteins pictured as
diffusing through the nuclear membrane into the
cytoplasm. RNA was considered to provide a tern
plate for protein synthesis. While the fact that at
hours the nuclear RNA has the highest specific
activity of the nucleic acids in the various cell
fractions
whereas
the highest
specific
activity
for
protein is in the microsomes does not necessarily
disprove this attractive hypothesis, at least it re
quires further explanation from its proponents.
Caspersson's theory must also be examined in the
light of two other lines of research. Abrams (1) has
shown that, whereas nucleic acid synthesis is in
hibited by x-radiation,
protein synthesis is not.
Brachet and Chantrenne
(8) have apparently
demonstrated
normal
that
protein
synthesis
rates in enucleated
takes place at
Acetabularia
mediter
ranea. Although an increased RNA content of grow
ing tissues is well established,
and is confirmed
in
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1953 American Association for Cancer Research.
@0o
Caiwer Research
the present work, the isotope experiments, which
reflect a more dynamic picture, show that there is
not the synchronization
of nuclear RNA and pro
tein
synthesis
that
the
Caspersson
theory
would
require. Similar conclusions have been reached by
Eliasson et al. (18).
The circumstance that the pool sizes of the acid
soluble phosphorus and uncombined glycine are of
the same order of magnitude lends some degree of
justification to a direct comparison of the incor
poration of glycine and phosphorus into the nu
cleic acid nucleotides. The finding that the C'4 and
@$2in
the
individual
punine
nucleotides
of
DNA
have the same specific activity suggests very
strongly that both precursors are incorporated
into some intermediate
at the same time and at
the same rate. In other words, DNA synthesis in
volves a process whereby the synthesis of the pu
nine and
phosphorus
moieties
of the
nucleotide
molecule proceed at the same rate, since the pool
sizes
@
@
of the
intermediates
are similar
with
respect
to both precursors.
Because we had previously reported ratios in
liver of RNA :DNA punines of about
: 1 for in
corporation
of glycine-@-C―, whereas ratios of
10:1 to 100:1 have been reported for p32, we con
eluded that “atleast two pathways of DNA syn
thesis exist. One involves the incorporation of pre
formed adenine together with desoxynibose and
phosphorus into the nucleic acid. The other and
perhaps preponderant
route involves the incon
poration of small molecule precursors of punines
into a skeleton to which the desoxyribose and
phosphorus have already been affixed― (ES). Now
that glycine and phosphorus have been studied
simultaneously
and are incorporated
into the
DNA to the same extent, with respect to both
specific and total nadioactivities, the former con
cept requires modification. Rather, as is clearly
shown by the experimental
data, the difference
between the incorporation
of the two precursors
lies in the RNA and not in the DNA. In the RNA
nucleotides of normal liver, the specific activity of
the
@32
in the nucleotides is @0times higher at
hours than that of the purine C'4. Therefore, the
process of incorporation of the two precursors into
RNA nucleotides must take place in such a way
that the phosphorus is incorporated
into a pre
cursor pooi at a time earlier than is the glycine. If
the glycine were also in equilibrium with some
intermediate
in the process, a further reduction of
C'4 specific activity would occur.
Strong experimental support for these concepts
has been provided independently
by Payne et al.
(88). They have studied in separate experiments
the incorporation
of p32, g1ycine-@-C'4, formate
C'4, and adenine-4,6-C'4
into nucleic acids of
mouse liven. They observed that the incorporation
at 4 hours of all these substances into DNA was
remarkably
similar. They also showed that the
ratio of cytoplasmic
RNA to DNA was much
higher for adenine and phosphorus than for for
mate, which had previously been shown to behave
similarly to glycine. Thus, in both these respects,
the results of their study and ours are in agree
ment. Furst and Brown (@0) found a RNA :DNA
ratio of 60 : 1 with the use of adenine and of 3:1
with glycine. Both labeled precursors were ad
ministered simultaneously.
They interpreted this,
as we had, to mean that there are two mecha
nisms of DNA synthesis. It appears likely that
adenine and phosphorus behave in the same fash
ion, and that their results are also due to a differ
ence in the behavior of the RNA, rather than in
the DNA. Since many steps must occur in the
biosynthesis of any substances as complex as nu
cleic acids, it would appear fruitless to continue
talking in terms of one or two mechanisms.
In the
experiments
of Furst and Brown (@0), the incor
poration of the two compounds into DNA is diffi
cult to compare, since different doses of the pre
cursors were used, no information was available
about pool sizes, and one compound was labeled
with N'5, a stable isotope, the other with radio
carbon. Thus, although at first sight their data
might seem to be in disagreement with ours, in the
absence of further information there is no reason
to believe that their study, that of Payne et al.
(38), and ours are in any way incompatible.
As a basis for further experimentation,
we have
been led to formulate a very tentative scheme of
nucleic acid biosynthesis in normal liver, which is
shown in Chart 18. This scheme is restricted to
nucleic acid adenine nucleotides, since the inter
relationships between adenine, guanine, and their
various precursors is unclean at the present time.
However, when such information becomes avail
able, it may easily be included in the scheme,
should it still have merit at that time.
One of the important
features
of the scheme
is
that DNA and RNA do not share common inter
mediates except for the distal precursors, glycine
and phosphorus. This seems plausible, in view of
the fact that the two parts of the molecule studied
here are connected in the case of DNA with des
oxyribose,
and
in RNA
with
ribose.
There
is at
present no evidence that the two sugars can be
intenconverLed. In the biosynthesis of RNA we en
visage the immediate formation of ribose-5-phos
phate, from @32
of high specific activity. This corn
pound then combines with an as yet unknown pre
cursor of nitrogen-9, and with forrnate (the pro
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1953 American Association for Cancer Research.
TYNER et aL—Radioactivity
in Nucleic
cursor of canbon-8) to give the “glycinelesspre
cursor― nibotide. This substance, which may only
be a transitory and unstable intermediate,
is then
thought to combine with glycine to give the nibo
tide of 4-amino-5-imidazolecanboxamide.
The gly
cine, utilized in this process, would have a lower
specific activity than the phosphorus, because it
would have been diluted in the course of the time
lag in the production
of the imidazolecanbox
amide nibotide. It should be pointed out that the
specific activity changes of the precursors at early
times are extremely rapid, and that only a small
time interval would probably be necessary to ac
count for the twentyfold difference in the specific
activities of the @32
and C'4 in the punine nucleo
@01
Acids and Proteins
tide, rather than the niboside, as a key intermedi
ate is in agreement with in vitro experiments of
Greenberg (@1) and of Schulman and his col
laborators (45, 46). Although the formulation of
these intermediates
in the scheme is consistent
with the work of others as well as with the present
investigation, direct proof of its accuracy can only
be obtained by the actual isolation and identifica
tion of the intermediates following the simultane
ous administration
of @32
and glycine or formate.
Such studies are now in progress, and should pro
vide additional information
on the question of
whether the 3' or 5' phosphates are the inter
mediates in nucleic acid biosynthesis.
In the scheme of DNA adenine nucleotide bio
DNA
I
Adenine ;± Adenine-desoxyribose-PO4
I
Imidazole carboxamide-desoxyribose-P04
I
I
Glycine —.4Glycine Pool
P04 Pool @—
P04
@I.
Ribose-5-PO4
Glycineless
precursor-ribose-P0@
Imidasole carboxamide-ribose-P04
1@1
Adenine-ribose-PO4 (AMP)
Adenine ;:±
RNA
CHART
18.—Tentative
scheme
of nucleic
acid
tides. This difference would be considerably en
hanced if the glycine to imidazolecanboxamide
re
action were reversible, which would result in the
further dilution of the glycine. The next inter
mediate is pictured as the punine nucleotide, which
is in agreement
with
the in vitro work of LePage
(3@), who has studied nucleic acid purine synthesis
in Ehnlich ascites tumor cells. The participation of
the imidazolecarboxamide
nibotide in inosinic acid
synthesis
in pigeon
liven extracts
has been demon
strated by Schulman and Buchanan
(45) and
Schulman et al. (46).
It appears that an important consequence of
this work is that whereas the ribotide is pictured
as being an intermediate, the riboside is not. If the
latter type of compound were an intermediate, we
would expect to find the specific activity of the
punine-C'4 to be higher than the P―, because it
would be removed from the precursor pool at an
earlier time than the phosphorus. This is contrary
to our observations. The participation
of the nibo
biosynthesis
with
respect
to
adenylic
acid
synthesis the glycine and phosphorus moieties link
up with the desoxynibose at the same time, and
this time must
be similar
to that
of the glycine
of
RNA, since the specific activities are of the same
order of magnitude. One suggestion to account for
this is that desoxynibose-5-phosphate
is formed,
the phosphorus migrates to give desoxynibose-1phosphate, and in the course of condensation with
the “glycinelessprecursor― this phosphate is lost.
The sugar is then rephosphorylated
with more
dilute
@32
at about the same time at which the
glycine is incorporated. It is a possible consequence
of this hypothesis that the desoxyniboside may be
an intermediate.
We have discussed previously the fact that the
tumor behaves qualitatively similarly to but quan
titatively different from liver (49). One conclusion
drawn at that time from a similarity of the rate of
turnover of adenine and guanine of DNA and
RNA was that “at
least the two purines of nucleic
acids must be degraded and resynthesized
to
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1953 American Association for Cancer Research.
Cancer Research
gether
in an organized
fashion― (49). This conclu
sion has been greatly strengthened in the present
study by a consideration of the total radioactivity
in
the
individual
RNA
nucleotides
shown
in
Charts 16 and 17. It is particularly evident in the
case of tumor (Chart 16) that the total
@32
incor
porated into the RNA molecule depends very
closely upon the nucleotide composition. It thus
appears
specific
that the rates of synthesis,
activity
of the individual
and, hence,
nucleotides,
the
are
controlled by the requirement
for these nucleo
tides as dictated by the composition of the nucleic
acid molecule. In the case of nongrowing liver
(Chart17),thecorrespondence
isnotsocloseas
that of tumor, but the fact that there is any cor
respondence at all between the total radioactivity
and nucleotide composition indicates to us that
there must be an actual synthesis of new RNA
molecules and not an independent turnover of the
individual nucleotides. This conclusion is in agree
ment with our earlier work (49) and with those of
Abrams (s), Payne et al. (38), and Eliasson et al.
(18).
Work now continuing along these lines involves
the isolation of intermediates
in the nucleic acid
nucleotide biosynthesis, and investigations of the
intramolecular
distribution of isotopes in the bio
synthesis of nucleic acids.
SUM@MARY
1. Glycine-@-C'4 and
@32
were simultaneously
injected intrapenitoneally
into normal rats and
others bearing multiple Flexner-Jobling carcinoma
transplants.
The animals were sacrificed after
various time intervals, and the livers and tumors
were fractionated by differential centnifugation in
to nuclear, mitochondnial, microsomal, and super
natant fractions. From each fraction the nucleic
acids were isolated, hydrolyzed, and the nucico
tides were separated by ion-exchange chromatog
raphy. The specific activity of the nucleotides
with
respect
to
@82
was determined,
the punine
nu
cleotides were hydrolyzed, and the C'4 specific ac
tivity of the punines was measured.
@.
From
the data
on the acid-soluble
phosphorus
and uncombined glycine, it was concluded that the
metabolic pool sizes of these are similar.
S. At early time intervals the order of decreasing
specific activity of both isotopes in the RNA of
cell fractions
was nuclei, supernatant,
micro
somes, and mitochondnia.
4. At early times the C'4 specific activity of the
proteins
was highest
in the microsomal
fraction.
The relationship of this finding to those for the
RNA has been discussed.
5. The specific activity of the DNA and RNA
nucleotides
in tumors
was higher
at all times than
in the corresponding liver nucleotides.
6. The specific activities of the @32
and C'4 in the
punine nucleotides from DNA were similar, mdi
eating that both precursors are incorporated into
some intermediate at the same time.
7. The
specific
activity
of the
@32
in the RNA
punine nucleotides of normal liver was considena
bly higher than that of the C―.This indicates that
the phosphorus is incorporated into an intermedi
ate at an earlier time than is the glycine.
8. The total radioactivities in the RNA nucleo
tides
of all cell fractions
are directly
proportional
to the total quantity of the nucleotides present.
This suggests that the composition of the nucleic
acid determines
the amount of synthesis and,
further, that the RNA molecule is synthesized as a
whole from the component nucleotide precursors.
9. The nucleic acid metabolism of tumors ap
pears to be qualitatively
similar, but quantita
tively
more rapid
than
that
of liver.
10. The systemic effect of a tumor on the metab
olism of liver is manifested
in an increased
specific
and total radioactivity
of the DNA nucleotides in
the livers of tumor-bearing animals as compared to
those
of normal
liver.
11. A tentative
thesis
scheme of nucleic acid biosyn
with respect
to adenylic
acid has been pro
posed in order to account for these findings. The
significance of this tentative
scheme has been
discussed.
REFERENCES
1. A@nai&s,R. Effect of X-Rays on Nucleic Acid and Protein
2.
Synthesis. Arch. Biochem., 30:90-90, 1951.
. Some Factors Influencing Nucleic Acid Purine
Renewal in the Rat. Ibid., 33:486—47, 1951.
S. B@utwuss,C. P., and HusEasT, R A. The Intracellular
Heterogeneity of Pentose Nucleic Acid as Evidenced by
the Incorporation of Radiophosphorus. Arch. Biochem.,
29:7—26,
1950.
4. Ban@ruu,C. P.; Husany, R. A.; and Vznwnro, H. Studies
on the Uptake of P@ by Mouse Mammary Carcinoma.
Cancer Research,
12:246,
1952.
5. Bzumzxs, M. Untersuchungen an isolierten Zell mid
Gewebsbestandteilen.
Kalbsherzmuskels.
I. Isolierung von Zeilkernen
des
Ztschr. Physiol. Chem., 209:59-68,
1952.
6. Bonsoox, H.; Dnasy, C. L.; HAAGEN-SMIT,
A. J.; KaiGu
LY, G.; and Lowy, P. H. Metabolism
of C―-Labeled Gly
cine, i.-Histidine,i@-Inucine,and ar.Lysine.J. BioLChem.,
187:889-48,
7. Boui@iona,
1950.
P.; Mowrmium,
J.; and [email protected], L. Cinétique
de l'echange du phosphore radioactif dane les ribonucléo
tides du foie de rat. Compt. rend., 234:565—68, 1952.
8. BnaciiET, J., and Ciwmiinara,
Nucleated and Non-Nucleated
mediierranea Studied
168:950,1951.
with
H. Protein Synthesis in
Halves of Ace4abularia
Carbon-14-Dioxide.
Nature,
9. Baowi@r,G. B.; PETERMANN,
M. L.; and FURST,S. S. The
Incorporation
of Adenine into Pentose and Desoxypentose
Nucleic Acids. J. Biol. Chem., 174: 1048-44, 1948,
10. Bnuiu,
A. M.; Tnacy,
M. M.; and Comi, W. E. Nucleic
Acids of Rat Liver and Hepatoma: Their Metabolic Turn
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1953 American Association for Cancer Research.
TYNER
over in Relation
to Growth.
et al.—Radwactivity
in Nucleic
J. Biol. Chem., 155:619—35,
1944.
11. BUSCH,H.; HURLBERT,R. B.; and Porrmt, V. R. Anion
Exchange Chromatography of Acids of the Citric Acid
Cycle. J. Biol. Chem., 196:717—27,1952.
12. CASPERSSON,
T. 0. Cell Growth and Cell Function, pp.
101—9.
New York: W. W. Norton & Co., 1950.
15. Comir, W. E. The Anion-Exchange Separation of Ribonu
cleotides. J. Am. Chem. Soc., 72: 1471—78,
1950.
14. DAVIDSON, J. N. In: Isotopes in Biochemistry,
pp. 175-83.
London: J. & A. Churchill, Ltd., 1951.
15. DAVIDSON,
J. N.; MchnoE, W. M.; and Swrn,
R. M. S.
The Uptake of P@by Ribonucleotides in Liver Cell Frac
tions. Biochem.
J., 49:xxxvi—vii, 1951.
16. DOUNCE,A. L. Enzyme Studies on Isolated Cell Nuclei of
Rat Liver. J. Biol. Chem., 147:685-98,
1945.
17. Dotmrci, A. L.; TISHEOFF,G. H.; Baiura@rr,S. R.; and
FREER, R. M. Free Amino
of Cell Nuclei Isolated
Technic.
J. Gen.
Acid and Nucleic
by a Modification
Physiol.,
33:629-42,
Acids
55. LEPAGE,G. A., and HEIDHLBERGER,
C. Incorporation of
Glycine-2-C―into Proteins and Nucleic Acids of the Rat.
J. Biol. Chem., 188:593—602,
1951.
34. LsPAOE, G. A.; Po!rrmi, V. R.; Buscii, H.; HnxDzi@
BEROER,
C.;
Implants
in
and
Hum@nunT,
Fed
and
R.
Fasted
B.
Growth
Rats.
of Carcinoma
Cancer
Research,
12:153—57,
1952.
55. Ln―n.H,
J. A., and Bumna, G. C. The Enzymatic Degrada
tion of Thymonucleic Acid. I. The Preparation of Oligonu
cleotides. J. Biol. Chem., 188:695—704, 1951.
56. M@tnsHAx,A. Evidence for a Nuclear Precursor of Ribo
and DesoxyribonucleicAcid. J. Cell. & Comp. Physiol.,
32:381—406, 1948.
37. Nsrr, R. J. In BARRON, E. S. G. (ed.), Modern Trends in
Physiology
and Biochemistry.
New York: Academic
Press,
1952.
38. PAvwr@,A. H.; KELLY,L. S.; and JONSR,H. B. The In
corporation
Acid Content
of the Behrens'
@03
and Proteins
of Formate-C14,
Glycine-2-C―, Adenine-4,6-
C'4 and Phosphate-P's into Nucleic Acids. Cancer Re
search, 12:666—70, 1952.
1950.
18. Eia@tssoN, H. A.; HAMMARSTEN,E.; REICHARD,P.;
AQUIST,S.; Thonnu@,B.; and EHRENSVXRD,
G. Turnover
Rates during Formation of Proteins and Polynucleotides
in Regenerating
Tissues. Acta Chem. Scandinav., 5:431—
44, 1951.
19. ELWYN,D., and SpnnisoN, D. B. The Extensive Synthesis
of the Methyl Group of Thymine in the Adult Itat. J. Am.
Chem. Soc.,72:5317—18,
1950.
20. FURST, S. S., and Baow@i, G. B. On the Role of Glycine and
Adenine as Precuiuors of Nucleic Acid Purines. J. Biol.
Chem., 183:289—47, 1951.
39. PAYNE,A. H.; KELLY,L. S.; and Wiirrsi, M. R. Effect of
Neoplastic
Tissue
on the
Turnover
of Liver
Nucleic
Acids. Cancer Research, 12:65—68,1952.
40. Po@rrnit,V. R.; RECHNAGEL,
R. 0.; and HTJRLBERT,
R. B.
Intracellular Enzyme Distribution; Interpretations and
Significance.
Fed. Proc., 10:646—55, 1951.
41. RHDDY,D. V. N., and Cminc@o, L. R. Effect of Tram
planted Tumors on Nucleic Acid Content
sues.Fed. Proc.,10:256,1951.
of Mouse Tis
42. REICHARD,P. Purine and Pyrimidine Turnover of Ribonu
cleic Acid from Fractionated
Rat Liver Cytoplasm.
Acta
21. Gn@mramiG,G. R. De NovoSynthesis of Hypoxanthine via
Chem. Scandinav., 4:861—65, 1950.
Inosine-5-Phosphate and Inosine. J. Biol. Chem., 190:611— 43. Scm@umna, W. C. Phosphorus Compounds in Animal
51,1951.
22. HAMMARSTEN,
E., and Hsvnsv, G. Rate of Renewal of
Ribo- and Desoxyribonucleic Acids. Acta Physiol. Scan
dinav., 11:885—43,1946.
28. HEIDELBERGER, C., and LEPAGH, G. A. Incorporation
of
Glycine-2-C―into Purines of Pentose Nucleic Acid and
Desoxyribose Nucleic Acid. Proc. Soc. Exper. Biol. &
Med.,76:464—65,
1951.
24. Ho@rciixjss, R. D. The Quantitative
Separation of Purines,
Pyrimidines, and Nucleosides by Paper Chromatography.
J.Biol.
Chem.,175:515—52,
1948.
25. Hui@m@, T. Incorporation
in Vivo of N@'-Labeled Glycine
into Liver Fractions of Newly Hatched Chicks. Exper.
Cell. Research, 1:376—81, 1950.
26. Hiiti@mr,T.; SLAUTTERBACK,
D.; and Wiussi,
poration
in Vivo of Labeled Nitrogen
G. Incor
and Phosphorus
into
the Ribonucleic Acid of Chick Liver Cytoplasm. Exper.
Cell. Research, 2:696—99, 1951.
27. Htmi.nmtT, R. B., and Porrnit,
V. R. A Survey
of the
Metabolism of Orotic Acid in the Rat. J. Biol. Chem.,
195:257—70,
1952.
28. HiiusT, R. 0.; Lrrmu,
Enzymatic
Degradation
J. A.; and Bumua,
G. C. The
of Thymonucleic
Acid. H. The
Hydrolysis of Oligonucleotides. J. Biol. Chem., 188:705—
15,1951.
29. JizNzu,
R., and Sz@s@utz,
D. Relations between the Rate
Tissues. I. Extraction and Estimation of Desoxypentosenu
cleic Acid and of Pentosenucleic Acid. J. Biol. Chem.,
161:293—303, 1945.
44. ScHNEIDER,W. C., and HoounooM, G. H. Intracellular
Distribution
of Enzymes.
V. Further
Studies on the Dis
tribution of Cytochrome c in Rat Liver Homogenates. J.
Biol. Chem., 183:125—28, 1950.
45. SCHULMAN,M. P., and [email protected], J. M. Biosynthesis of
the Purines. II. Metabolism
of 4-Amino-5-Imidazole
carboxamide in Pigeon Liver. J. Biol. Chem., 196:513—21,
1952.
46. Scnuiat&rr, M. P.; Soni@in,J. C.; and Buciwwi, J. M.
Biosynthesis of the Purines. I. Hypoxanthine Formation
in Pigeon Liver Homogenates and Extracts. J. Biol. Chem.,
196:499—512,
1952.
47. SIEK.EYITZ,P. Uptake of Radioactive Alanine in Vitro into
the Proteins of Rat Liver Fractions.
J. Biol. Chem.,
195:549—65,
1952.
48. TOTrER, J. R.; VoLxin, E.; and CARTnn,C. E. Incorpora
tion of Isotopic Formate
into the Nucleotides
of Ribo and
Desoxyribosenucleic Acids. J. Am. Chem. Soc., 73:1521—
22, 1951.
49. TYNER,E. P.; Humm@nmiomt,C.; and LEPAGE,G. A. In
Vise Studies
on Incorporation
teins and Nucleic
Acid Purines.
of Glycine-2-C'4
Cancer
Research,
into Pro
12:158—
64,1952.
50. VOLKIN,E., and CARTER,C. E. The Preparationand
of Renewal and the Intracellular Localization of Ribonu
Properties
of Mammalian
Ribonucleic
Acids. J. Am.
cleic Acid. Arch. Biochem., 26:54—67,1950.
Chem. Soc., 73:1516—19, 1951.
80. [email protected], E. B. Turnover of Proteins of Cell Fractions of
Adult Rat Liver in Vivo. Fed. Proc., 10:206, 1951.
51.
. The Incorporation of Isotopic Phosphate in the
Mononucleotides of Liver Nucleic Acids. J. Am. Chem.
51. LAIRD,A. K.; NYGAARD,0.; and Ris, H. Separation of
Soc.,
73:1519—20,
1951.
Rat Liver Mitochondria into Two Morphologically and
52. VOLKIN,E.; Kiims, J. X.; and Com'@,W. E. The Prepara
Biochemically Distinct Subfractions. Cancer Research,
12:279-77, 1952.
tion of Desoxynucleotides.
J. Am. Chem. Soc., 73:1533-36,
1951.
52. LEPAGE, G. A. In Vitro Incorporation of Glycine-2-C'4
into Purines and Proteins. Cancer Research, 13:178—85, 53. WYATT,G. R. Purine and Pyrimidine Composition of
1953.
Desoxypentosenucleic Acid. Biochem. J., 48:584—90,1951.
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1953 American Association for Cancer Research.
Intracellular Distribution of Radioactivity in Nucleic Acid
Nucleotides and Proteins Following Simultaneous
Administration of P32 and Glycine-2-C14
Evelyn Pease Tyner, Charles Heidelberger and G. A. LePage
Cancer Res 1953;13:186-203.
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