Download Effects of 6-Thioguanine on RNA Biosynthesis in Regenerating Rat

[CANCER
RESEARCH
37, 1876-1882, June 1977]
Effects of 6-Thioguanine on RNA Biosynthesis in Regenerating
Rat Liver1
Christine K. Carrlco2 and Alan C. Sartorelli
Departmentof Pharmacologyand Sectionof DevelopmentalTherapeutics,ComprehensiveCancerCenter,YaleUniversitySchoolof Medicine,NewHaven,
Connecticut06510
SUMMARY
6-Thioguanine, at a dose of 40 mg/kg body weight, was
administered to rats at 12 hr after partial hepatectomy; 6 hr
later, liver polysomes and cell sap were isolated and utilized
to measure the effects of this antimetabolite on protein
synthesis in vitro. When radioactive leucine was used to
label peptides synthesized in vitro, no difference was ob
served between polyacrylamide gradient gel scans of sys
tems derived from control regenerating liver and those from
6-thioguanine-treated regenerating liver. However, when
radioactive tyrosine was used as the tracer to monitor syn
thesized peptides, a depression in the 30,000-molecular
weight region of scans of products synthesized in systems
derived from 6-thioguanine-treated regenerating liver was
observed. Recombination experiments showed this effect to
be due to the pobysome component of the system. When
equal amounts of polyadenybic acid-containing RNA from 6thioguanine-treated
or control regenerating liver were
added to a wheat germ in vitro protein-synthesizing system,
pobyacrybamidegel scans of the products synthesized in the
presence of radioactive tynosine showed that more peptides
were synthesized from polyadenylic acid-containing RNA
from 6-thioguanine-treated rats than from control polyade
nylic acid-containing RNA. That this phenomenon might be
the result of incorporation of the analog into RNA was
shown by the finding that all types of RNA contained 6thioguanine, with the greatest concentration occurring in
polyadenylic acid-containing RNA.
the synthesis of other kinds of RNA was apparently unaf
fected by the punine antimetabobite. These effects were
initiated in the G1 phase of the cell cycle and appeared to
constitute a new metabolic lesion(s) created by 6-TG; thus,
it was of importance
to attempt
to localize
more precisely
the site of action of the pumine antimetabolite on these
metabolic events. Earlier work by Wheeler et a!. (20) has
shown that 6-TG acts in the G, phase of the cell cycle of
H.Ep.2 cells to delay the G1- to S-phase transition; the
biochemical alteration responsible for this action is un
known but would appear to be unrelated to incorporation of
the analog into DNA.
GTP is required at several stages of the translational
process; therefore, protein synthesis would appear to be a
particularly vulnerable site for the action of a guanine ana
bog. Roy et a!. (14), however, demonstrated that when 6-TG
tniphosphate was substituted for GTP in an in vitro amino
acid-incorporating system, not only did the thiopurine nu
cleotide not inhibit the reaction, but in contrast, it was
found to partially substitute for the natural substrate. Gray
and Rachmeler (6) reported that 6-TG was incorporated into
Escherichia coli tRNA and that this incorporation appar
ently affected the amino acid acceptor activities of some of
the tRNA's.
6-TG could also be envisioned to interfere with the syn
thesis of certain proteins either by being incorporated into
the specific mRNA molecules for these proteins or by affect
ing the synthesis of the specific mRNA's themselves. The
punine antimetabolite has been shown to be incorporated
into both RNA and DNA (8, 9, 15, 17), but it is the incorpora
tion into DNA to which cytotoxicity has been attributed (see,
INTRODUCTION
e.g., Ref. 10).
The experiments described in this paper show that in vitro
The previous paper of this series (4) has showrtthat 6-TG3 translation of certain groups of peptides is altered in cell
has a major inhibitory effect on the partial hepatectomy
free systems derived from 6-TG-treated regenerating liver
induced synthesis of enzymes required to support DNA and that the basis of this effect resides in the polysomes.
replication in regenerating rat liver without altering the mate Furthermore, 6-TG is preferentially incorporated into
of total protein synthesis in vivo. These findings were ac
poly(A)@ RNA, and this 6-TG-containing RNA is more effi
companied by a depression of the synthesis of poly(A)
cient in directing in vitro translation than is poIy(A)@RNA
containing RNA in 6-TG-tneated regenerating liver, whereas without 6-TG. These effects are observed at a time when
DNA synthesis
I Support
was
provided
by
Grants
CA-02817
and
CA-16354
from
the
Na
and 6-TG incorporation
into these macro
molecules in regenerating liver are minimal.
tional Cancer Institute, USPHS.
2 Recipient
of
Training
Grant
GM-0059
from
the
General
Medical
Sciences,
USPHS. The work described in this report is a portion of the work presented
in partial fulfillment of the requirements for the Ph.D. degree.
3 The
abbreviations
used
are:
6-TG,
6-thioguanine;
poly(A),
polyadenylic
acid; poIy(A)@RNA, polyadenylic acid-containing RNA; SDS, sodium dodecyl
sulfate; poly(A) RNA, polyadenylic acid-lacking RNA; PCA, perchloric acid.
Received January 14, 1977; accepted March 21, 1977.
1876
MATERIALS AND METHODS
Wheat germ, pynuvate kinase, phosphoenolpyruvate,
RNase A, dithiothreitob, creatine phosphate, creatine phos
phokinase, N-2-hydnoxyethybpipenazine-N'-2-ethanesulfonic
CANCER RESEARCH VOL. 37
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6-TG Action on RNA Biosynthesis
acid, and carbonic anhydrase were obtained from Sigma of Roberts and Paterson (13). The peak turbid fractions
Chemical Co., St. Louis, Mo.; acrylamide and N,N'-methyl
were pooled, rapidly frozen in 0.5-mb batches, and stored at
enebisacrybamide were purchased from Eastman Kodak —70°.
Wheat germ in vitro protein synthesis assays were
Co., Rochester, N. Y. CelIex-D anion-exchange resin was performed in a total volume of 0.5 ml, containing 0.2 ml
obtained from Bio-Rad Laboratories, Richmond, Calif.; wheat germ S-30 fraction, 20 mM N-2-hydroxyethylpipera
[4,5-3H (N)]Ieucine and elemental 355 were obtained from zine-N'-2-ethanesulfonic acid, pH 7.6 (adjusted with KOH),
New England Nuclear, Boston, Mass.; [2,6-3H (N)Jtyrosmne 2 mi@idithiothreitob, 1 mM ATP, 20 @M
GTP, 8 mM creatine
was purchased from ICN Chemical Radioisotope Division, phosphate, 3 mM magnesium acetate, 100 mM KCI, 30 @M
Irvine, Calif. fledisobv HP was obtained from Beckman In
cold amino acid mix without tynosine, 50 @Ci[3,5-3H
(N)]tyrosine, 20 /hg creatine phosphokinase, and 50 @g
struments, Inc., Fullerton, Calif. 6-TG was the generous gift
of Dr. George H. Hitchings of Bummoughs-Wellcome Re
poIy(A)@RNA isolated as described previously (4). The meac
search Laboratories, Research Triangle Park, N. C.
tion was started by the addition of the 5-30 fraction and was
Male Sprague-Dawley rats (Charles Riven Breeding Labo
allowed to proceed for 30 mm at 25°,after which time 10 j.@g
ratonies, Wilmington, Mass.), weighing between 170 and 200 RNase and EDTA to a final concentration of 10 mM were
g, were housed routinely over corn cob bedding, and alter
added. After incubation for 15 mm at 37°,the peptides were
nating periods of 12 hr dark and 12 hr light were main
precipitated by the addition of 2 volumes of 80% acetone at
tamed. Before surgery, Purina rat chow and water were 00. The precipitate
was
collected
by
centnifugation
and
washed 3 times with 80% acetone, after which it was sus
available ad libitum. Partial hepatectomies were performed
pended in 0.0625 M Tmis-HCI,pH 6.8-2% SDS-10% glycerol
under light ether anesthesia between 8:30 and 10:30 a.m.
according to the method of Higgins and Anderson (7). 6-TG 0.1 M dithiothneitol-0.01% bromphenol blue for poly
was dissolved in 0.9% NaCI solution with the aid of dilute acrylamide gel electrophonesis on gradient pobyacrylamide
NaOH as described previously (4) and was injected i.p. at 12 gels.
Linear (4 to 30%) gradient acrybamide gels were prepared
hr after partial hepatectomy, at a dose of 40 mg/kg body
weight; control animals received an equal volume of 0.9% by using an apparatus described by Caton and Goldstein (5).
Chamber 1 contained 51.45 g acrylamide and 1.05 g N,N'
NaCI solution.
methylenebisacrybamide in 150 ml of 0.375 M Tnis-HCI, pH
The rat liver in vitro protein-synthesizing system utilized
was a composite of those described by Ragnotti et a!. (12) 8.8-0.1% (w/w) SDS, and Chambem2 contained 5.72 g acryl
amide and 0.28 g N,N'-methylenebisacrybamide in 150 ml of
and Atkins et a!. (2). Partially hepatectomized and normal
animals were sacrificed by decapitation, and their livers the above Tnis buffer. Immediately before starting the gra
were excised, minced, and homogenized in 2 volumes of dient former, 0.15 ml N ,N ,N' ,N―-tetnamethylethylenedia
mine and 0.075 g of ammonium pemsulfate were added to
0.25 M sucrose-50 mM Tnis-HCI, pH 7.8-25 mM KCI-6 mM
MgSO4. This homogenate was centrifuged at 12,000 x g ton each chamber and mixed well. The gels were allowed to
10 mm, and the supemnatant solution was mecentnifuged at polymerize overnight at room temperature, after which time
145,000 x g for 2 hr. The resulting pellet, resuspended in they were cut free from the excess gel encasing the glass
incubation buffer (0.15 M sucnose-35 mM Tnis-HCI, pH 7.8-25 tubes. Gels prepared in this fashion could be stored under
buffer in the cold for several weeks.
mM KCI-10 mM MgSO4), was used as the source of poly
somes in the cell-free system. The 145,000 x g supernatant
Before electrophoresis of peptides, gels were subjected
solution was passed through a Sephadex G-25 coarse cob to preelectrophoresis for 45 mm at 2 ma/gel. Samples were
umn equilibrated with incubation buffer. The initial frac
applied in a volume of 50 @b
and subjected to ebectrophore
tions of this column were used as the source of amino acid
sis in a Buchlen Polyanalyst water-jacketed electrophomesis
free cell sap for the cell-free incubations.
cell at 2 ma/gel, until the bromphenol blue marker reached
The cell-free incubation system consisted of 2 to 4 ml cell the bottom of the tube (approximately 4.5 to 5 hr). Electro
sap; 0.5 to 2.0 ml polysomes (containing 10 to 20 mg RNA) phonesis buffer was 0.025 M Tnis-0.193 M glycmne, pH 8.3,
in incubation buffer; 175 @g
pyruvate kinase pen ml, 4 ml of containing 0.1% SDS. Gels were chopped into 2-mm seg
a stock solution consisting of 2 mM ATP, 0.25 mM GTP, and ments directly from the electrophoresis tubes with a Gibson
Aliquogel fractionator, and radioactivity in the gel sections
10 mM phosphoenolpyruvate,
and 35 @Ci
[3,5-3H (N)]tyrosine
was determined in Beckman Redisolv HP using a Packard
(24.6 Ci/mmole)
or 100
@Ci[3,5-3H (N)Jleucmne (40 Ci!
mmole) in a total volume of 10 ml. One ml of a 300 mM cold
Tni-Cambliquid scintillation spectrometer.
amino acid mix lacking only the radioactive amino acid
Protein markers were run each time a series of gels was
utilized was also added to the incubation flask. The reaction
subjected to electrophoresis. The marker proteins used
was started by the addition of polysomes and allowed to were cytochnome c, RNase A, and carbonic anhydmase,
proceed at 37°for 1 hm,after which time 200 @g
RNase A in possessing molecularweightsofl.17
x 10@,1.37 x 10@,and
10 mM EDTA were added. After 30 mm at 37°,the proteins
3.40 x 10@,respectively. Marker gels were stained with 0.1%
Coomassie blue in 7.5% acetic acid-50% methanol and de
were precipitated at 0°with 80% acetone. The precipitate
was dissolved in sample buffer for linear (4 to 30%) gradient stained in 7.5% acetic acid-50% methanol. Following de
polyacrylamide gel ebectrophonesis (0.0625 M Tmis-HCI, pH staining, the marker gels were scanned at 600 nm in a
Beckman DU spectrophotometer equipped with a Gilford
6.8-2% SDS-10% glycerol-0.1 M dithiothreitol-0.01%
bromo
Model 2140 linear transport accessory utilizing a scanning
phenol blue).
aperture of 2.4 x 0.05 mm and a scanning mateof 1 cm/mm.
The 5-30 fraction for the wheat germ in vitro protein
6-[35S]TG was synthesized according to the method of
synthesizing system was prepared according to the method
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1877
C. K. Carrico and A. C. Sartorelli
Morávek and Nejedb@(11). 6-TG was dried at 110°in a
vacuum overP205forseveralhr.To a suspensionof500 mg
(2.99 moles) of 6-TG in 200 ml of dry pymidine, which had
been refluxing for3 hn, were added 10 mCi (1 to 20 mCi/mg)
of elemental 355in benzene solution. The vial containing the
@
RESULTS
Typical gradient polyacrylamide gel profiles of peptides
synthesized in the rat liver in vitro protein-synthesizing sys
tem as measured by the incorporation of [3H]leucine are
shown in Chart 1. No difference was detected in the pep
355 was rinsed
5 to 7 times
with
pynidine,
and these
tides synthesized in systems derived from either 18-hr par
washes were added to the refluxing solution. The suspen
sion was refluxed (protected by a calcium chloride tube) for tially hepatectomized control livers or from partially hepa
3.5 hr. The pynidine was removed in a vacuum. The residue tectomized , 6-TG-treated livers.
However, when tyrosine was substituted for leucine as the
was resuspended in tobuene, and this was removed in a
vacuum to eliminate all pynidine from the product. The dry radioactive amino acid used in the rat liver in vitro protein
synthesizing system, the peptide profiles on polyacrylamide
residue was recrystallized from 480 ml of water after deco
Ionization with charcoal and concentration of the filtrate to gels shown in Chart 2 were obtained; in these studies as
150 ml. The crystals were collected after 48 hr of mefrigera well as for those described in Chart 1 using leucine as the
tion and dried with air and acetone. The yield was 115 mg measure of proteins synthesized, both the polysomes and
the cell sap were derived from the same liver. The data show
(23%) of 6-[35S]TG (3 mCi/mmole).
For measurement of the incorporation of 6-TG into RNA, that in one particular region (3 to 5 cm), corresponding to
matswere given 6-[35S]TG at 40 mg/kg body weight 12 hr approximately M.W. 30,000, the synthesis of peptides was
significantly depressed in the in vitro protein-synthesizing
after partial hepatectomy. Six hr later the matswere sacni
ficed by decapitation, and their livers were excised and system derived from 6-TG-tmeated regenerating liver, rela
homogenized in 3 volumes of 0.32 M sucmose-3mM MgCI2. tive to that from control regenerating liver. The average area
Nuclear poly(A)@ RNA and poby(A) RNA were isolated as under the affected peaks from nine 6-TG-denived systems
described previously (4). Cytoplasmic RNA was isolated was 52% of the average control area from 8 scans (p <
0.005). The reason for the amino acid specificity toward
from the 700 x g supernatant solution after centnifugation
to remove the mitochondnia and precipitation with 95% leucine and tyrosine in the action of 6-TG is unknown;
presumably, it reflects the amino acid composition of the
ethanob-2% potassium acetate at —20°
overnight. The pre
cipitate
was dissolved
in10 mM sodium acetate,
pH 5.1-0.14 newly synthesized peptides, since the profiles of peptides
synthesized in the presence of tyrosine and leucine differ
M NaCI-0.01%
sodium dextran sulfate-0.3%
SDS, and the
RNA was extracted and separated into its poly(A)@ and markedly with respect to peak position and height.
poly(A) components as described (4). The absorbance at
II'
260 and 280 nm was determined for each fraction. RNA
CONTROL
fractions were then hydrolyzed at 37°for 1 hr in 0.3 N KOH.
300
After neutralization with PCA, the hydrolysates were applied
to Celbex-D anion-exchange columns (30 x 5 mm), equili
E
200
brated with 0.02 M Tnis-HCI, pH 8.0. Following elution with
z
11 ml of 0.02 M Tmis-HCI,pH 8.0, to remove 6-TG present as
0
the free base, nucleotides were eluted with 2 ml of 1 N HCI,
i: 100
‘4
and radioactivity therein was determined using Beckman
0
Redisolv HP in a Packard Tni-Carb liquid scintillation spec
trometer.
0
C.,
6-TG
z
The presence of 6-TG nucleotide in DNA was determined
after an i.p. injection of 6-[35S]TGat 40 mg/kg body weight.
w
z
Purified nuclei were isolated from 18- and 24-hr regenerat
200
ing liver as described previously (4). The 50,000 x g pellet
w
was resuspended in 1 ml of 0.32 M sucmose-3mM MgCI2 and
100
precipitated with 4.5 volumes of 0.5 N PCA. Following cen
tnifugation at 270 x g for 10 mm, the pellet was dissolved in
0
4 ml of 0.3 N KOH and heated at 37°for 1 hr to hydrolyze the
0
1
2
3
4
5
6
7
8
RNA. Following the addition of 2.5 ml of 1.2 N PCA and
DISTANCE MIGRATED (Cm)
centnifugation at 270 x g for 10 mm, the pellet, which
Chart 1. Gradient polyacrylamide gel electrophoretic scans of products
consisted of DNA and protein, was dispersed in 4 ml of 0.5 N derived
from the nat liven in vitro protein-synthesizing system using
(‘I
Q.
—
@
300
-J
@
I
PCA and heated to 70°for 20 mm. The DNA hydrolysate
was
chilled and centrifuged at 270 x g for 10 mm. Abiquots of the
supemnatant solution were taken for measurement of DNA
concentration by the method of Burton (3) using calf thy
mus DNA as a standard. The remainder of the supernatant
solutions were neutralized with KOH and chromatographed
on Ceblex-D anion-exchange resin; then, deoxynucleotide
fractions collected, and radioactivity therein was deter
mined as described above for RNA samples.
1878
I
I
I
I
I
rH]Ieucine as the tracer. 6-TG ( 0) was injected i.p. into rats 12 hr after partial
hepatectomy at a dose of 40 mg/kg body weight. The control group (•)
received an equivalent volume of 0.9% NaCI solution. At 18 hr after partial
hepatectomy the rats were sacrificed, and the liver polysomes and cell sap
were isolated. The polysomes and cell sap were added to an in vitro system
containing pyruvate kinase, [3H]leucine, ATP, GTP, phosphoenolpyruvate,
and an amino acid mixture lacking leucine as described under “Materials
and
Methods.―Following incubation, peptides were subjected to electrophoresis
on 4 to 30% linear gradient polyacrylamide gels. The gels were divided into 2mm sections, and the radioactivity in each segment was determined. Car
bonic anhydrase (molecular weight of 34,000) used as a standard migrated
4.1 cm.
CANCER RESEARCH VOL. 37
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6-TG Action on RNA Biosynthesis
300
@
CONTROL
800
200
z
600
0
i: 100
E
‘4
400
U
0
Q.
z
8300
z
I‘4
0
6- TG
“a
@
0
5.
200
U)
200
0
0
C.,
z
@-
‘ii
z
100
800
U,
0
0
0
1
2
3
4
5
6
DISTANCE MIGRATED (Cm)
7
8
Chant 2. Gradient polyacrylamide gel electrophoretic scans of products
derived from the rat liver in vitro protein-synthesizing system using
@H]tyrosineas the tracer. The data were obtained by procedures identical to
those described in Chart 1 except that (3Hjtyrosine was used instead of
leucine as the radioactive amino acid, and the cold amino acid mix lacked
tyrosine instead of leucine.
In an effort to determine whether the polysome compo
nent or the cell sap component was responsible for the
observed decrease by 6-TG in the synthesis of a peptide or
peptides containing tyrosine, an experiment was performed
in which polysomes from 18-hr control regenerating liver
were added to an in vitro protein-synthesizing system con
taming cell sap from 18-hr 6-TG-treated regenerating liver. A
polyacrylamide gel profile from this experiment is shown in
Chart 3 (bottom). Profiles of the product(s) synthesized in
the presence of polysomes from control regenerating liver
and cell sap from drug-treated regenerating liver indicate
that the 30,000-M.W. region was present apparently in unab
tered form (compare to control profile; Chart 3, top).
The converse experiment, that is, the use of polysomes
from 18-hr 6-TG-treated regenerating liver with cell sap from
18-hr control regenerating liven, was performed (Chart 4,
top); the 6-TG gel profiles clearly demonstrated the induced
inhibitory effect (Chart 4). From these experiments, it was
concluded that the decrease in tyrosine-containing pep
tides synthesized in the in vitro system derived from 6-TG
treated regenerating liver was due to the polysome compo
nent rather than to the cell sap component.
To determine whether the observed effect might be due to
a decrease in the quantity of specific mRNA's in polysomes
from the livers of 6-TG-treated animals, a wheat germ in
vitro protein-synthesizing system was used which allowed
the addition of an equal quantity of poIy(A)@RNA from the
livers of 6-TG-treated or control rats to the incubation mix.
The peptides synthesized in vitro were separated on gra
dient polyacrybamide gels, and representative duplicate ra
dioactivity profiles of gels from preparations nun with con
trol and 6-TG poly(A)@RNA are shown in Chart 5. The areas
under the affected peaks from 6-TG-denived systems were
30% greater than the areas under the peaks from control
systems (p < 0.005), indicating that more total peptides
were being synthesized from poIy(A)@RNA from 6-TG
I-
600
400
200
2
3
DISTANCE
4
5
MIGRATED
6
7
8
(cm)
Chart 3. Gradient polyacrylamide gel electrophoretic scans of products
from a recombined rat liver in vitro protein-synthesizing system in which
polysomes from untreated partially hepatectomized animals and cell sap
from 6-TG-treated rats were used. The data were obtained by procedures
identical to those described in Chart 2, except that in both cases polysomes
were obtained from control partially hepatectomized rat liver and cell sap
from either 6-TG-treated or untreated regenerating liven.
treated rats than from control poby(A)@RNA.
That this effect might be due to the presence of 6-TG in
mRNA was shown by the finding that 6-TG was incorporated
into all species of RNA as a nucleotide (Table 1). Much more
6-TG was incorporated
into poIy(A)@ RNA than was incorpo
rated into poly(A) RNA under these conditions. This effect
was particularly striking in the nucleus where poby(A)@
RNA
incorporated 50 times more 6-TG than did poly(A) RNA. 6TG was also incorporated
into the DNA of regenerating
rat
liver. As expected, at 18 hr after partial hepatectomy, the
liver DNA contained only 31 ±8 pmoles of 6-TG in nucleo
tide form per mg of DNA. At 24 hr after partial hepatectomy,
however, during peak synthesis of DNA in regenerating
liver, DNA contained 494 ±55 pmoles of 6-TG nucleotide
per mg of DNA. Quantities of 6-TG equal to or greater than
those incorporated into the nucleic acids were associated
with these macromolecules, particularly poby(A)@RNA,
demonstrating the need for isolation of 6-TG nucleotide
from both DNA and RNA to insure that the antimetabolite
was incorporated as part of the structure of these mole
cubes. The dose of 6-TG used in these studies would appear
to be reasonable, since the degree of incorporation into
RNA correlates well with the cytotoxic
level reported by Tidd
and Paterson (18).
DISCUSSION
The previous report (4) demonstrated that 6-TG had major
effects on the 1st wave of partial hepatectomy-induced en
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1879
C. K. Carrico and A. C. Sartorelli
J
800
•
I
I
I
CONTROL CELL SAP
6-TG POLYSOMES
6OO@
I
-
U
-
z 300
E
0
I-
‘4
0.
U
0
a.
z
0
0
200
z
“a
z 100
U
0
a.
0
C.)
z
“a
z
U,
6-TG CELL SAP
6-TG POLYSOMES
0
800
.
-
600
-
-
400
-
U)
>-
I-
0
I-
0
a.
C.)
200
z 300
0
I-.
‘4
DISTANCE
MIGRATED
(cm)
Chart 4. Gradient polyacrylamide gel electrophoretic scans of products
from a recombined rat liver in vitro protein-synthesizing system in which
polysomes from 6-TG-treated, partially hepatectomized animals and cell sap
from untreated control rats were used. The data were obtained by proce
dunes identical to those described in Chart 2, except that in both cases
polysomes were obtained from 6-TG-treated, partially hepatectomized rat
liver and cell sap from either 6-TG treated- or untreated regenerating liver.
0
a. 200
0
U
z
z“a100
U)
0
>-
zyme synthesis and DNA replication in regenerating matliver
and that the biosynthesis of poly(A)@RNA was depressed in
6-TG-treated regenerating liver before the synthesis of DNA.
To provide evidence for a connection between these 2 phe
nomena, an in vitro protein-synthesizing system was used
which allowed a close inspection of the translational proc
ess. A rat liver system was chosen for the initial studies
because evidence is available which indicates that the initi
ation factors are species and, possibly, tissue specific (1,
19). The 2 major separable components
of this sytem are the
polysomes and the cell sap. The polysome portion consists
of nibosomes, mRNA, bound tRNA, and bound factors. The
cell sap component contains the majority of the tRNA, the
ammnoacyl-tRNAsynthetases, and the free factors (i.e., EF1,
EF2, and R).
Gray and Rachmelen (6) have reported that the acceptor
activity of tymosyb-tRNAin E. co!i is susceptible to the action
of 6-TG and have equated this sensitivity with the incorpora
tion of the analog into these tRNA molecules. For this
reason, radioactive tyrosine was chosen as one of the Ia
beled precursors to be used in the in vitro protemn-synthesiz
ing system. In addition, since leucine had been used to
assess the effects of 6-TG on total protein synthesis in vivo
(4), radioactive beucine was also used in the in vitro protein
synthesizing system. The profiles of peptides labeled in the
presence of radioactive tyrosine and those labeled in the
presence of radioactive leucine differed markedly, implying
that different peptides (on groups of peptides) are being
1880
l@
0
2
4
6
DISTANCE
8
2
MIGRATED
4
6
8
(cm)
Chant 5. Gradient polyacrylamide gel electrophoretic scans of products
derived from the wheat germ in vitro protein-synthesizing system. 6-TG was
injected i.p. into rats 12 hr after partial hepatectomy at a dose of 40 mg/kg
body weight. The control group received an equivalent volume of 0.9% NaCI
solution. At 18 hr after partial hepatectomy, the rats were sacrificed, and the
cytoplasmic poly(A)@RNA was isolated from their livers. Equal amounts of
poby(A)@RNA from control or 6-TG-treated regenerating livers were added to
a wheat germ in vitro protein-synthesizing system containing wheat germ 530 fraction, dithiothreitol, ATP, GTP, creatine phosphate, magnesium ace
tate, KCI, creatine phosphokinase, [‘H]tyrosine,and a cold amino acid mix
lacking tyrosine as described under “Materials
and Methods.―The synthe
sized products were processed as described in Chart 1.
represented. These peptides appear to contain quite differ
ent amounts of these 2 amino acids, and some of the pep
tides high in tyrosine content are sensitive to the action of 6TG, whereas those containing more beucine are not. Thus,
in profiles of tyrosine-containing peptides synthesized in
vitro from 6-TG-denived systems, a decreased tyrosine in
corporation occurred into 1 particular region with no appar
ent shifting of the position of the peaks in that region. This
result bendssupport to the concept, derived from studies in
viVo (4), that the protein synthetic machinery per se is not
altered by 6-TG, since at beast 1 group of peptides (i.e.,
those high in leucine content) are apparently synthesized in
the same fashion in the in vitro protein-synthesizing sys
tems derived from the livers of 6-TG-tneated and control
mats.
CANCER RESEARCHVOL. 37
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6-TG Action on RNA Biosynthesis
1Incorporation
Table
tional system allows the addition of equivalent amounts of
poly(A)@ RNA from control and 6-TG-treated regenerating
(3mCi/mmole)
12 hr after partial hepatectomy,rats were given 6@[US]TG livers, no difference would be expected in the polyacryl
ratswere
at a dose of 40 mg/kg body weight. Six hr later,
amide gel profiles of peptides synthesized in vitro if the sole
andhydrolyzed
sacrificed, and their liver RNA fractions were isolated
in 0.3 N KOH.The neutralized hydrolysateswere chro effect of 6-TG were to decrease the amount of mRNA syn
thesized in vivo. That the functional ability of the mRNA
matographedon Cellex-Danion-exchangecolumns to separate
6-TG
from its nucleotide, as described under “Materials
and Meth from the livers of 6-TG-treated animals to be translated in
ods.―RNA
vitro was altered is evidenced by the fact that significantly
more peptides high in tyrosine content were synthesized
pmoles/mgbNucleus
source
RNAfractiona
from 6-TG-containing poly(A)@RNA than from an equivalent
(4)CPoIy(A)@
PoIy(A)
142 ±57
amount of control poby(A)@RNA. This finding might be the
(4)Cytoplasm
7203 ±2603
result of incorporation of the punine antimetabolite into
(6)Poby(A)@
Poby(A)
37 ±11
mRNA in a manner that allows more efficient translation.
124 ±50 (5)
Since both the initiator and the terminator codons contain a
a Poly(A)@
and poly(A)
RNA were
isolated
from
total
nuclear
and
guanine moiety, one possibility is that substitution of 1 or
cytoplasmic
RNA on pobyuridylate-Sephanose
as described previ
both of these guanine residues by a 6-TG molecule might
ousby(4).
drastically alter either the mateof initiation or termination.
b Incorporation
of 6-TG
is expressed
as pmobes
6-TG
occurring
as the nucleotide per mg of RNA±S.E.
The fact that 6-TG-containing poly(A)@RNA is translated
C Numbers
in parentheses,
number
of animals
in each
detenmi
more efficiently in the wheat germ system cannot be extrap
nation.
olated to the liver in vitro translational system because it is
doubtful whether the products synthesized in the 2 systems
are identical; thus, the polyacrylamide gel profiles of these 2
The polysome component of the in vitro protein-synthe
sets of products differ. Since different mRNA molecules
sizing machinery appears to be responsible for the inhibi
probably incorporate different amounts of 6-TG and in dif
tory effect of 6-TG seen in profiles of high-tyrosine-contain
fementboci within the molecule, the ultimate effects on pep
ing peptides synthesized in vitro. This implies that the nibo
tide synthesis that one observes are probably the result of a
somes, the mRNA, or the bound factors are being affected
by 6-TG, rather than the tRNA as reported for E. co!i by Gray combination of actions by 6-TG on mRNA.
and Rachmeler (6). Although 6-TG, incorporated into cer
The data presented in this paper are supportive of the
tam tRNA molecules, may alter their amino acid acceptor
postulated mechanism presented in the earlier report (4) in
activity, this effect does not appear to be significant in the in which 6-TG, given before the onset of S phase, interferes
vitro protein-synthesizing system used in these expeni
with the synthesis of certain proteins by depressing the
ments. Data presented in the previous paper (4), on the synthesis of their mRNA's. In addition, 6-TG is presumably
effects of 6-TG on the synthesis of poIy(A)@RNA in regenem incorporated into the mRNA's for these enzymes; use of the
ating liver, indicate that the most likely site of action of 6-TG wheat germ system has allowed the detection of an altered
on the polysomes responsible for inhibition of protein syn
efficiency in the mateof translation of these 6-TG-containing
thesis in vitro is the mRNA.
mRNA molecules. The occurrence of such an effect in vivo
Since the time at which 6-TG was administered to mats might result in either an altered amount of enzyme on en
corresponded to a time after partial hepatectomy when a zyme molecules with an impaired function on both.
2nd burst of RNA synthesis occurs (see, e.g., Ref. 16), it
This entire sequence of events occurs before the 1st wave
seemed reasonable to assume that at least some of the of mitosis after partial hepatectomy. The initiating events,
analog was incorporated into this newly synthesized RNA. which may be related, occur in G1, before the onset of DNA
That this assumption was indeed true was demonstrated by synthesis, and therefore, the action of 6-TG on these meta
the finding that 6-TG was incorporated into RNA at this time bolic events would appear to be divorced from the incompo
to a significant extent. Under the conditions used, the ration of the punine antimetabolite into DNA.
poly(A)@RNA incorporated significantly more 6-TG per mg
of RNA than did the poly(A) RNA. In addition, the poly(A)@
ACKNOWLEDGMENTS
RNA fraction exhibited a greater response to the action of 6TG in terms of diminished incorporation of radioactive on
capable assistance of Vow-Mci Huang and Paula A. Wilson is grate
otic acid and decreased A260than did the poly(A) RNA (4). fullyTheacknowledged.
In the regenerating liven, like other cell systems, signifi
cantly more 6-TG was found in the RNA fraction as com
REFERENCES
pared to the DNA fraction, although 6-TG nucleotide was
found in both DNA and RNA. Although the method used to
1. Arstein, H. R. v. Competition between Eukaryotic Messenger RNAS in
measure 6-TG incorporation into nucleic acids eliminates
Cell-free Protein Synthesis. Acta Biol. Med. Ger., 33: 971-978, 1974.
the contribution of binding of the base or nucleoside to the
2. Atkins, J. F., Lewis, J. B., Anderson, C. ‘N.,
and Gesteland, R. F.
liverAt
of 6-(35SJTGinto RNA of regenerating
DNA and RNA, it does not differentiate
between
labeled
drug present in intennucleotide linkage from any 6-TG nu
cleotide bound to the nucleic acid fractions.
The results obtained by use of a wheat germ in vitro
protein-synthesizing system indicate a possible additional
effect of 6-TG on mRNA. Because the wheat germ tnansla
Enhanced Differential Synthesis of Proteins in a Mammalian Cell-free
System by Addition of Polyamines. J. Biol. Chem., 250: 5688-5695, 1975.
3. Burton, K. Determination of DNA Concentration with Diphenylamine.
Methods Enzymol., 12 (Part B): 163-166, 1968.
4. Carrico, C. K., and Sartorelli, A. C. Effects of 6-Thioguanine on Macromo
lecular Events in Regenerating Rat Liver. Cancer Res., 37: 1868-1875,
1977.
5. Caton, J. E., and Goldstein, G. Electrophoresis of Ribonucleic Acids on
JUNE 1977
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1977 American Association for Cancer Research.
1881
C. K. Carrico and A. C. Sartore!li
Polyacrylamide Gel Gradients. Anal. Biochem., 42: 14-20, 1971.
6. Gray, P. N., and Rachmeler, M. The Effects of 5-Fluorouracil and 6Thioguanine Incorporation on the Amino Acid Acceptor Activity of Each
erichia coli tRNA. Biochim. Biophys. Acta, 138: 432-435, 1967.
7.
8.
9.
10.
11.
Higgins,
G.
M.,
and
Anderson,
R. M.
Experimental
Pathology
of the
Liver. I. Restoration of the Liver of the White Rat Following Partial
Surgical Removal. Arch. Pathol., 12: 186-202, 1931.
Kwan, S-W., Kwan, S-P., and Mandel, H. G. The Incorporation of 6Thioguanine into RNA Fractions and Its Effect on RNA and Protein
Biosynthesis in Mouse Sarcoma 180 Ascites Cells. Cancer Res., 33: 950955, 1973.
LePage, G. A. Incorporation of 6-Thioguanine into Nucleic Acids. Cancer
Res., 20: 403-408, 1960.
LePage, G. A., and Jones, M. Further Studies on the Mechanism of
Action of 6-Thioguanine. Cancer Res., 21: 1590-1594, 1961.
Morávek,J., and NejedI@,Z. Labelling of 6-Mercaptopunine and 6-Mer
captopurine Riboside by an Exchange Reaction with Elementary Sulfur
355•
Chem.
Ind.,
p. 530,
15.
16.
17.
18.
1960.
12. Ragnotti, G., Lawford, G. R., and Campbell, P. N. Biosynthesis of Micro
somal Nicotinamide-Adenine Dinucleotide Phosphate-Cytochnome c Re
ductase by Membrane-bound and Free Polysomes from Rat Liver. Bio
chem. J., 112: 139—147,
1969.
13. Roberts, B. E., and Paterson, B. M. Efficient Translation of Tobacco
Mosaic virus RNA and Rabbit Globin 95 RNA in a Cell-free System from
1882
14.
19.
20.
Commercial Wheat Germ. Proc. NatI. Acad. Sci. U. S., 70: 2330-2334,
1973.
Roy, J. K., Kvam, D. C., DahI, J. L., and Parks, R. E., Jr. Effect of
Tniphosphate Nucleosides of 8-Azaguanine, 6-Thioguanine, and 6-Mer
captopunine on Amino Acid Incorporation in Vitro into Microsomal Pro
tein. J. Biol. Chem., 236: 1158-1 162, 1961.
Sartorelli, A. C., LePage, G. A., and Moore, E. C. Metabolic Effects of 6Thioguanine. I. Studies on Thioguanine-resistant and Sensitive Ehrlich
Ascites Cells. Cancer Res., 18: 1232-1239, 1958.
Thaler, M. M., and villee, C. A. Template Activities in Normal, Regenerat
ing and Developing Rat Liver Chromatin. Proc. NatI. Acad. Sci. U. S., 58:
2055-2062, 1967.
Tidd, D. M., and Paterson, A. R. P. Distinction between Inhibition of
Punine Nucleotide Synthesis and the Delayed Cytotoxic Reaction of 6Mercaptopunine. Cancer Res., 34: 733—737,
1974.
Tidd, D. M., and Paterson, A. R. P. A Biochemical Mechanism for the
Delayed Cytotoxic Reaction of 6-Mercaptopunine. Cancer Res., 34: 738746, 1974.
Weissbach, H., and Brot, N. The Role of Protein Factors in the Biosyn
thesis of Proteins. Cell, 2: 137-144, 1974.
Wheeler, G. P., Bowdon, B. J., Adamson, D. J., and Vail, M. H. Compani
son of the Effects of Several Inhibitors of the Synthesis of Nucleic Acids
upon the Viability and Progression through the Cell Cycle of Cultured
H.Ep. No. 2 Cells. Cancer Res., 32: 2661-2669, 1972.
CANCER RESEARCH VOL. 37
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1977 American Association for Cancer Research.
Effects of 6-Thioguanine on RNA Biosynthesis in Regenerating
Rat Liver
Christine K. Carrico and Alan C. Sartorelli
Cancer Res 1977;37:1876-1882.
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