Download 3-Deazaguanine: Inhibition of Initiation of Translation in L1210 Cells1

[CANCER RESEARCH 42. 4039-4044,
0008-5472/82/0042-OOOOS02.00
October 1982]
3-Deazaguanine: Inhibition of Initiation of Translation in L1210 Cells1
Rachel S. Rivest,2 David Irwin, and H. George Mandel3
Departments of Pharmacology
¡R.S. R. H. G. M.] and Biochemistry [D. I.], The George Washington University Medical Center, Washington. D. C. 20037
ABSTRACT
In L1210 cells in culture, 3-deazaguanine (DG), a relatively
new purine analog, was found to inhibit DNA and protein
synthesis but not total RNA synthesis. The effect of the drug
on protein synthesis was therefore further examined. Polyadenylic acid-containing RNA synthesis was not decreased by
DG treatment, suggesting that the inhibition of protein synthesis
was a function of an alteration in the process of translation. DG
altered the polyribosome sedimentation profile in a dose-de
pendent manner, increasing the numbers of monosomes and
smaller polysomes and decreasing the number of larger polysomes. The nascent polypeptides in DG-treated cells were
labeled with [3H]leucine, and the increased number of monosomes was not associated with a proportionate amount of
[3H]leucine when compared to the polysomes. This indicated
that the monosomes had not been derived directly from the
breakdown of active polysomes. The shift in the polysome
profile was reversed by cycloheximide, suggesting that DG
inhibited the initiation of translation. This was confirmed by the
demonstration of the inhibition by DG of the formation of the
43S preinitiation complex. The inhibition of the initiation of
protein synthesis by DG may contribute to the antitumor actions
of this new purine analog.
INTRODUCTION
DG" is a relatively new purine analog with interesting antitumor, antiviral, and antibacterial activity (1, 3, 7, 9, 20, 22, 23).
Although most of the still limited number of studies suggested
a primary effect of DG on DNA synthesis (8, 20, 23) as well as
alterations in the formation of purine intermediates (26), our
investigations pointed to an inhibitory effect on protein synthe
sis as well (15, 16). Since 2 other growth-inhibitory
purine
analogs also are known to affect the synthesis of proteins
(reviewed in Ref. 17), we have investigated in greater detail the
inhibition of protein synthesis by DG in L1210 cells in culture.
The reversal by cycloheximide of the shifts in the polyribosome
sedimentation profiles from DG-treated cells suggested that
DG inhibited the initiation of translation. This was confirmed by
a reduction in the binding of 35S-formylated methionine tRNA
' This investigation was supported by USPHS Grants CA-02978 and CA27553 and Training Grant 5-T-32-CA-09223.
awarded by the National Cancer
Institute, Department of Health & Human Services.
2 National Cancer Institute Trainee (Grant 5-T-32-CA-09223).
Present ad
dress: Department of Pharmacology, Yale University School of Medicine, New
Haven, Conn. 06510. From a dissertation presented to the Department of
Pharmacology, The Graduate School of Arts and Sciences, The George Wash
ington University, in partial fulfillment of the requirements for the Ph.D.
3 To whom requests for reprints should be addressed.
* The abbreviations used are: DG, 3-deazaguanine:
RPMI, Roswell Park
Memorial Institute Tissue Culture Medium; HBSS, Hanks' balanced salt solution;
poly(A)* RNA, polyadenylic acid-containing RNA; poly(A)" RNA, polyadenylic
acid-lacking RNA; TCA, trichloroacetic acid; deaza-GTP, 3-deazaguanosine 5'triphosphate.
Received November 17, 1981 ; accepted July 1, 1982.
OCTOBER
1982
to the ribosomal initiation complex in L1210 cells treated with
DG.
MATERIALS AND METHODS
Materials. DG was a gift from ICN Pharmaceuticals, Irvine, Calif. Cell
culture supplies (RPMI 1640, fetal calf serum, streptomycin, penicillin
G, and HBSS) were received from Grand Island Biological Co., Grand
Island, N. Y. RPMI 1630 without methionine was from the Media Unit,
NIH, Bethesda, Md. [metf7y/-3H]Thymidine (20 Ci/mmol), [5-3H]uridine
(28 Ci/mmol), [3H]adenosine (35 Ci/mmol), ['"C]uridine
(50 mCi/
mmol), i_-[4,5-3H]leucine (50 Ci/mmol), Aguasol, and Omnifluor were
from New England Nuclear, Boston, Mass. i_-{35S]Methionine (1200
Ci/mmol)
was obtained from Amersham/Searle
Corp., Arlington
Heights, III. Nonidet P-40 was from Particle Data Laboratories, Elmhurst, III. Type 2 and type 3 oligodeoxythymidylic
acid-cellulose was
purchased from Collaborative Research, Waltham, Mass.
Cell Culture. The L1210 tumor line, obtained from Dr. Kurt Kohn,
National Cancer Institute, Bethesda, Md., was maintained in RPMI
1640 supplemented with 10% dialyzed fetal calf serum. The medium
contained 1 x 105 units penicillin G and 0.1 g streptomycin sulfate per
liter of medium. Cells were maintained in suspension at 37° in a
humidified incubator with a 5% C(X>atmosphere.
Measurement of Viability. Viability of L1210 cells was measured by
the colony formation assay (2). Cells were exposed for 5 hr to 5, 10,
15, 20, 30, 40, 50, or 500 ¡IMDG dissolved in the medium. The cells
were washed with fresh medium and diluted in RPMI 1640 containing
20% dialyzed fetal calf serum. Three ml of RPMI 1640:20% dialyzed
fetal calf serum containing 0.2% Noble agar at 44° were added to
L1210 cells in 2 ml of RPM11640:20% dialyzed fetal calf serum, mixed
thoroughly in a 15-ml tube, placed on ice for 2 to 4 min, and then
incubated upright at 37°.The colonies were counted visually 10 to 12
days later.
Measurement of Macromolecular Synthesis. The effect of DG on
DNA, RNA, and protein synthesis was determined by the method of
Mandel ef al. (11). The L1210 cells were exposed to 5, 50, or 500
fiM DG for 5 hr and then incubated with either [3H]thymidine, [3H]uridine, or [3H]leucine at 1 /iCi/ml. An equal volume of ice-cold 0.9%
NaCI solution was added to each sample to stop the reaction after 60
min, and the samples were put on ice. The cell suspensions were
filtered directly on Whatman GF/C filters wetted with 0.9% NaCI
solution and washed with 5 volumes of 0.9% NaCI solution, 10 volumes
of 0.2 N perchloric acid, and 5 volumes of 0.9% NaCI solution. All of
the filtering solutions were ice cold. The filters were dried and counted
in 0.4% Omnifluortoluene.
To determine the effect of DG on poly(A)+ and poly(Ar RNA synthe
sis, L1210 cells were exposed to 50 JJ.MDG for 5 hr and labeled during
the last hr of drug treatment with either [3H]adenosine (10 /iCi/ml) or
[14C]uridine (0.1 juCi/ml). The cells were chilled rapidly with ice-cold
HBSS, centrifuged, and lysed with gentle vortexing in isotonic buffer
(150 mw NaCI:10 mw Tris HCI, pH 7.4:10 mw MgCI2) containing 0.5%
Nonidet P-40, a nonionic detergent. The crude nuclei were pelleted by
centrifugation at 1000 x g for 5 min, and the supernatant was centri
fuged at 10,000 x g for 15 min to obtain a postmitochondrial
super
natant. The supernatant was diluted in 10 volumes of 0.1 M NaCI:0.2%
sodium dodecyl sulfate: 10 ITIM Tris, pH 9.0:10 ITIM EDTA and then
extracted several times with an equal volume of phenol and of chlorofornrisoamyl alcohol (96:4). The last extraction was done with chloro-
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R. S. Rivest et al.
form:isoamyl alcohol (96:4), and the RNA in the resultant aqueous
phase was precipitated with 2 volumes of 95% ethanol overnight at
-20°.
was added with mixing, followed by 0.11 volume of 40% formaldehyde
neutralized to pH 7 on the day of use. The solution was held on ice for
a minimum of 30 min and was then layered over 9-ml preformed CsCI
The pellet of RNA obtained after ethanol precipitation was resuspended in binding buffer (0.5 M NaCI:0.01 M Tris HCI, pH 7.5:0.5%
sodium dodecyl sulfate) and applied to an oligodeoxythymidylic
acid-
gradients, 1.35 to 1.60 g/ml. The gradients were centrifuged for 40 hr
at 27,000 rpm at 4°. Fractions were collected on ice, and 1 drop of
cellulose column [0.25 g each of type 2 and type 3 oligodeoxythymi
dylic acid-cellulose] equilibrated with binding buffer. After elution with
15 ml binding buffer to collect the poly(A)~ RNA, the poly(A)+ RNA
was eluted with 15 ml of eluting buffer (0.01 M Tris HCI, pH 7.5:0.5%
sodium dodecyl sulfate). Five M NaCI was added to the poly(A)* RNA
0.5% bovine serum albumin was added to each fraction as a carrier,
followed by 5 volumes of cold 20% TCA. The precipitate was collected
on Whatman GF/C filters and washed with 10% TCA, 5% TCA, water,
and 95% ethanol. The filters were dried, and radioactivity was mea
sured in 0.4% Omnifluortoluene.
fraction to make a final concentration of 0.1 M NaCI. Two volumes of
95% ethanol were added, the RNA was precipitated overnight at —20°, RESULTS
and the precipitate was collected by centrifugation at 10,000 x g for
20 min at -20°. The pellets were resuspended in 0.1 M NaCI:0.2%
sodium dodecyl sulfate:! 0 mM Tris, pH 9.0:10 mM EDTA, and the
aliquots were measured for radioactivity using Aquasol as the scintil
lation cocktail.
Polyribosome Sedimentation Profile Analysis. L1210 cells were
exposed to 10, 30, 50, 100, or 500 ,UMDG for 5 hr. The cells were
chilled rapidly with ice-cold HBSS, and the postmitochondrial
super
natant, isolated as described above, was layered over 35-ml 15 to 40%
linear sucrose gradients made in lysing buffer without the Nonidet P-
Viability and Macromolecular Synthesis. After treatment
with 50 or 500 /ÕMDG for 5 hr, the viability of L1210 cells
decreased to 25% of control. At such concentrations, DG did
not inhibit [3H]uridine incorporation into nucleic acids, whereas
[3H]thymidine and [3H]leucine incorporation was decreased to
near 50% of control values (Chart 1). Although total RNA
synthesis was not depressed by DG, the possibility of a de
crease in mRNA synthesis, which could explain the inhibition
of protein synthesis, needed to be excluded. Since this fraction
40. The gradients, centrifugea for 2.5 hr at 26,000 rpm in a SW 27
represents only a small proportion of RNA, such an effect might
rotor, were pumped through a flow cell, and the absorbance at 260 nm
have been masked in the previous studies. However, DG was
was recorded continuously.
shown to produce no significant effect on the synthesis of
To rule out the possibility that the shift in the polysome profile was
poly(A)+ RNA (mRNA), as measured by the incorporation of
due to fragmentation of mRNA, L1210 cells were exposed to 50 /¿M [3H]adenosine (Table 1). Since measurement of the incorpo
DG for 5 hr, and during the last 3 min of the incubation, the cells were
ration of this precursor into poly(A)+ RNA actually measured
pulse labeled with [3H]leucine (20 juCi/ml) to label the nascent polypeptide chains on the polysomes. The cells were chilled rapidly with
HBSS, and the polysomes were displayed on sucrose gradients as
described above. Fractions were collected, and the radioactivity was
determined.
The ability of cycloheximide to reverse the alterations in the polysome profile was determined by a modification of the method of
Reichman and Penman (14). L1210 cells were divided into 4 cultures,
of which 2 were control and 2 were exposed to 50 /¿M
DG for 5 hr. One
control and one DG culture were also exposed to cycloheximide (2 /ig/
ml) for the last 30 min of the incubation. The cells were chilled rapidly
with HBSS, and the polysomes were displayed on sucrose gradients
as described above.
Initiation Complex Formation Assay. The formation of the 43S
ribosomal preinitiation complex in drug-treated cells was determined
the rate of 2 processes, i.e., the synthesis of mRNA and the
addition of the poly(A)+ RNA tail, the RNA was labeled sepa
rately with [14C]uridine to accurately measure only the synthesis
of the body of the mRNA. Here again, the inhibition of protein
synthesis by DG could not be explained by a depression in
mRNA synthesis (Table 1).
Polyribosome Sedimentation Profile Analysis. The polyribosome sedimentation profile from L1210 cells exposed to 50
120
by a modification of the method of Henshaw (5, 6). L1210 cells were
labeled overnight with [3H]uridine (1 fiCi/ml) to uniformly label the RNA.
The cells were resuspended in fresh medium and exposed to 50 ¡IM
DG for 5 hr. The cells were centrifuged at 37°, and the pellet of 2 x
107 cells was resuspended in 0.5 ml of RPMI 1630 lacking methionine
but containing DG. The cells were preincubated at 37°for 2 min and
then labeled with [35S]methionine (200 ,uCi/ml) for 2 min. The reaction
was stopped with 10 volumes of ice-cold HBSS, and an aliquot was
removed to measure the incorporation of label into proteins by precip
itation (see below). The rest of the sample of cells was lysed, and the
postmitochondrial
supernatant was isolated as described previously.
The supernatant was layered over 12-ml linear 20 to 30% sucrose
gradients made in 100 mM KCI, 2 mM magnesium acetate, 20 mM
triethanolamine-HCI (pH 7.0), and 0.5 mM dithiothreitol. The gradients
were centrifuged at 25,000 rpm for 16 hr at 4°in a SW 41 rotor. The
10~5
10'*
10'3
absorbance of the gradients was monitored, and the fractions contain
ing the 40S to 45S ribosomal subunits were collected. Due to the poor
resolving power of sucrose gradients, 40S subunits isolated from these
gradients were contaminated by other ribonucleoprotein
species and
by [35S]methionine-labeled proteins.
Chart 1. Effect of DG on macromolecular synthesis and viability. Cells were
exposed to DG for 5 hr and then labeled for 1 hr with [3H]thymidine, [3H]uridine,
or [3H]leucine. Incorporation of the isotopes into the acid-insoluble fraction was
These species were separated further from each other with CsCI
density gradients. To the fraction isolated from the sucrose gradients,
0.1 volume of 0.11 M morpholinopropane
sulfonic acid-KOH (pH 7.0)
determined, and the values presented are the mean of triplicate samples. The
standard deviations were less than 2% of mean values. Top, effect of DG on cell
viability, determined by the colony formation assay for the various drug concen
trations.
4040
CONCENTRATION (M)
CANCER
RESEARCH
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VOL. 42
Inhibition of Initiation by DG
/IM DG for 5 hr showed increased numbers of monomers and
small polysomes and a decrease in the number of large polysomes (Chart 2). The decrease in the polysome fraction, i.e.,
that fraction of total ribosomes engaged in translation, was
dose-related (see Chart 2, inset), and the cytotoxic and polysomal effects of DG occurred at the same concentration range
(10 to 50 fiM DG).
One possible mechanism for this shift in the polysome profile
caused by DG was fragmentation of active mRNA during ex
traction. This possibility was examined by labeling the nascent
polypeptides of cells (treated with 50 fiM DG) with [3H]leucine.
The increased numbers of monosomes that resulted from DG
treatment were not associated with a proportionate amount of
[3H]leucine when compared to the amount of [3H]leucine as
sociated with the polysomes (Chart 3). This indicated that the
monosomes were nonfunctional and had not been derived
directly from breakdown of active polysomes.
This shift in the polysome profile caused by DG is similar to
that resulting from impaired initiation of translation (14). Cycloheximide at low concentrations is known to inhibit the elonga
tion step of translation without significantly affecting initiation,
whereas at high doses both steps are inhibited (25). The
slowing of elongation by low concentrations of cycloheximide
would be expected to counteract the effect on the polysome
Table 1
Effect of DG on polyW
Mean ±S.D.
1Not significantly
received 2 /ig cycloheximide per ml for the last 30 min of the
incubation, and the polysome profile of each of these samples
was determined.
In control cells exposed to cycloheximide, the polysome
sedimentation profile shows a reduction in the monosome peak
and an increase in all sizes of the polysomes (Chart 4). Cyclo1.5
1.0
10
c 0.5
o
to
o
l
UJ 0.4
5
IT
O
î
and poly(Ay
RNA synthesis
control)
control)
84.5 ±7.3
101.0 ±5.7
79.8 ±5.7
91.5 ±7.16
10
Top
20
Bottom
Chart 3. Effect of DG on labeling of nascent polypeptides. Cells were exposed
to 50 /IM DG for 5 hr and during the last 3 min were pulse labeled with [3HJleucine. The polysomes were displayed on 15 to 40% sucrose gradients, fractions
were collected, and radioactivities were determined.
different from poly(A)* values.
CONTROL
CONTROL*
VOLUME
CYCLOHEXIMIDE
(ml)
Chart 2. Effect of DG on the polyribosome sedimentation profile. Cells were
exposed to 50 JIM DG for 5 hr, and the polyribosomes were displayed on a 15 to
40% sucrose gradient.
. drug-treated cells;
, control cells. Inset, effect
of DG on the polysome fraction. The polysome fraction was calculated by
expressing the absorption of the polysomes (the area from dimers to bottom of
gradient) as a percentage of the total ribosomal absorption (monomers to bottom
of gradient). LD.»,,50% lethal dose.
1982
30
VOLUME (ML)
—
OCTOBER
S
0.2
L1210 cells, exposed to 50 JIM DG for 5 hr. were labeled during the last hr of
drug treatment with either [3H]adenosine or ("CJuridine. The RNA in the postmitochondrial supernatant was extracted and then separated into poly(A)* RNA
and poly(Ar RNA fractions on oligodeoxythymidylic
acid-cellulose columns. The
incorporation of the isotopes into RNA was measured.
poly(A)» RNA (% of
poly(A) RNA (% of
Label
[3H]Adenosine
['"CJUridine
profile of a drug which inhibits initiation, as was shown for 5azacytidine (14). The effects of a low concentration of cyclo
heximide (2 /¿g/ml)on the polysome profile following DG treat
ment were therefore examined. L1210 cells were divided into
4 cultures, 2 of which were untreated and 2 of which received
50 juM DG for hr. One control and one DG-treated culture
VOLUME (ml)
Chart 4. Effect of cycloheximide on the polyribosome sedimentation profile.
Cells were exposed to 2 fig cycloheximide per ml for 30 min, and the polysomes
were displayed on a 15 to 40% sucrose gradient.
4041
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R. S. Rivest et al.
heximide, which inhibited [3H]leucine incorporation into pro
teins by 80% (16), prevented the analog-induced shift towards
monosomes and returned the polysome fraction of DG-treated
cells back toward that of control cells (Chart 5). In addition,
although the relative size distribution of polysomes was not
altered by cycloheximide in control cells, in DG-treated cells
cycloheximide reversed the greater preponderance of smaller
polysomes and increased the fraction of larger polysomes. The
reversal by cycloheximide of the shift in the polysome sedi
mentation profile from DG-treated cells further suggested that
DG inhibited the initiation of translation.
Initiation Complex Formation. The results obtained by the
use of cycloheximide suggested but did not directly prove that
DG inhibited the initiation of translation. In the subsequent
experiments, formation of radiolabeled formylated methionine
tRNA (the initiator aminoacyl tRNA species) and incorporation
into the 43S and preinitiation complex served as the basis for
the direct assay of initiating activity in DG-treated cells. L1210
cells therefore were grown overnight with [3H]uridine to uni
The ratio of 35S to 3H counts associated
3000 - 1000
2000 o_
O
- 500
—
1000 -
formly label the RNA, were treated with 50 /XMDG for 5 hr, and
then were exposed for 2 min to [35S]methionine. The 43S
preinitiation complex thus contained the [35S]methionine, which
had condensed with the initiator methionyl-tRNA. The 40 to
45S region isolated from sucrose gradients was then further
displayed on CsCI gradients.
The results of the effects of DG on the formation of the 43S
preinitiation complex are shown in Chart 6. The various ribonucleoprotein species, shown in Chart 6, separated on the
CsCI gradients are, from left to right, the 60S ribosomal subunit,
the 80S ribosome and initiation complex, the 40S ribosomal
subunit, and the 43S preinitiation complex. The 35S label was
not associated with the first 3 species but was recovered
around Fraction 30, which represents the 43S preinitiation
complex, and around Fraction 40, which represents proteins.
with the 43S peak
decreased after DG treatment, indicating that DG inhibited the
formation of the 43S preinitiation complex.
These results, summarized in Table 2, show the agreement
between the decrease in the incorporation of [35S]methionine
10
20
30
SAMPLE NUMBER
40
3000 -
2000 -
O.
O
1000 -
20
30
SAMPLE NUMBER
DG
Chart 6. Effect of DG on the formation of the 43S formylated methionine tRNA
preinitiation complex. Cells labeled overnight with [3H]uridine were treated with
50 /IM DG for 5 hr and then were exposed for 2 min to [35S]methionine. The 40S
DG+CYCLOHEXIMIDE
subunits, isolated on sucrose gradients, were displayed on CsCI gradients.
Fractions were treated as described in "Materials and Methods." The major
tritium peaks represent, from left to right, the 60S, 80S, 40S (p = 1.52), and
43S (p = 1.44) ribonucleoprotein species, and the 35S activity was distributed
mainly in 2 peaks, representing the 43S initiation complex (p = 1.44) and
proteins (p = 1.30).
Table 2
Inhibition by DG of formation of 43S preinitiation complex and of total protein
synthesis
LI 210 cells, labeled overnight with [3H]uridine, were treated with 50 ^M DG
for 5 hr and then were exposed for 2 min to [35S]methionine. Aliquots were
removed to measure [35S]methionine incorporation into proteins. The 40 to 45S
region, isolated from sucrose gradients, was displayed on CsCI gradients. The
ratios of 35S to 3H cpm in the 43S preinitiation complex fractions of the CsCI
gradients were used as a measure of preinitiation
complex formation.
synthesis3 (%
Experiment1
20
10
VOLUME (ml)
of control)45
control)5133
30
Bottom
Chart 5. Effect of cycloheximide on polyribosome sedimentation profile alter
ations in DG-treated cells. Cells were exposed to 50 JIM DG for 5 hr and for the
last 30 min were also exposed to 2 ng cycloheximide per ml. Polysomes were
displayed on a 15 to 40% sucrose gradient.
4042
preinitiation com
plex formation6 (% of
3749
2
3
4443S
MeanProtein
* [35S]Methionine incorporation into TCA-insoluble
0 35Scpm/3H cpm; TCA-precipitable material.
CANCER
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45
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RESEARCH
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VOL. 42
Inhibition of initiation by DG
into total proteins and the diminished formation of the 43S
preinitiation complex. Protein synthesis decreased to 44% of
control, and the formation of the 43S preinitiation complex
decreased to 45% of control after treatment with 50 /¿M
DG.
DISCUSSION
DG inhibited protein synthesis and DMA synthesis in L1210
cells in culture, whereas RNA synthesis was not decreased.
The effect of DG on DMA synthesis and the lack of effect of DG
on RNA synthesis have been reported in L1210 and Chinese
hamster ovary cells (20, 23), and it has been concluded that
the effects of the drug on DNA synthesis were most closely
associated with cytotoxicity (8).
Although DG did not selectively decrease protein synthesis
in Chinese hamster ovary cells (20), the inhibition of protein
synthesis that we observed in L1210 cells correlated both with
the dose and length of drug exposure (15). In addition, the
inhibition of protein synthesis after treatment of the cells with
50 UM DG, as measured by the incorporation of [3H]leucine,
[3H]tyrosine, and [3H]lysine into the acid-insoluble fraction, was
60, 59, and 55% of control, respectively (16). In later experi
ments with [35S]methionine (see Table 2), a comparable inhi
bition of protein synthesis was seen. Since no selective inhibi
tion of any of the major cytoplasmic proteins from DG-treated
cells could be observed by polyacrylamide gel electrophoresis
(15) and since poly(A)+ RNA synhesis was not decreased by
DG treatment, this suggested that the inhibition of protein
synthesis was a function of an alteration in the process of
translation.
The shift in the polysome profile caused by DG was similar
to that resulting from impaired initiation of translation. The
possibility that the shift in the polysome profile was a result of
fragmentation of active mRNA during extraction was elimi
nated. The nascent polypeptides of cells treated with DG were
labeled with [3H]leucine, and the increased numbers of monosomes that resulted from DG treatment were not associated
with a proportionate increase in the amount of [3H]leucine when
compared to the polysemes. This indicated that the monomers
had not been derived directly from the breakdown of active
polysomes.
The slowing of elongation by low concentrations of cycloheximide would be expected to counteract the effect on the
polysome profile of a drug which inhibits initiation, as was
shown for 5-azacytidine (14). Treatment of control cells with
cycloheximide indicated an increase in the loading of all
mRNAs with ribosomes due to the decreased rate of elongation.
Once on the mRNA, each ribosome took a longer time to travel
along the message before it dissociated, leading to a uniform
increase in the amount of ribosomal RNA associated with each
size of mRNA. For DG-treated cells later exposed to cyclohex
imide, the shift from lighter to heavier polysomes suggested
that mRNAs had not been fully loaded with ribosomes in the
presence of DG. When ribosomes attached at substantially
lower rates than in controls because of a block in initiation, the
slowing of elongation by cycloheximide then allowed more time
for messages to become fully loaded with ribosomes.
These results, which suggested that DG inhibited the initia
tion of translation, were confirmed by the demonstration of the
inhibition of the formation of the 43S preinitiation complex. The
agreement between the decrease in the formation of the initi
OCTOBER
1982
ation complex and the decrease in protein synthesis suggests
that DG inhibits protein synthesis by inhibition of the initiation
step of translation. In addition, the inhibition of the 80S initiation
complex has also been demonstrated by us (17). However, the
precise mechanism for the inhibition of the formation of the
43S preinitiation complex has not yet been ascertained.
A block in translational initiation could result from interfer
ence in the role of GTP which is an essential requirement for
initiation. This is possible since DG needs to be metabolized to
its nucleotide form for activity (9, 21) and since DG interferes
with de novo purine biosynthesis (26). Therefore, the formation
of deaza-GTP or a reduction in GTP concentrations could lead
to an inhibition of initiation. The presence of deaza-GTP in
tumor cells following treatment with DG has been reported
(23), and direct measurements of DG-induced alterations of
the pools of purine nucleotides are pending. However, another
guanine analog, 8-azaguanine, has been shown by us to inhibit
initiation in a manner similar to that of DG (17, 18), and its
actions do not appear to be related to the levels of nucleotide
triphosphates (10, 13, 19, 27). Thus, it is doubtful that the
effect of DG in interfering in the initiation process is due to an
effect involving deaza-GTP or diminished levels of GTP.
DG could also block initiation by incorporation into any of
the RNA species so as to alter their structure and function. DG
was reported to be incorporated into nucleic acids (23), but
this incorporation did not inhibit the synthesis of RNA in our
system. There is no direct evidence to suggest that DG affects
the function of mRNA either through incorporation into the
body of the message or through interference with the 7-methylguanosinyl-5' cap modification of mRNA. However, 8-aza
guanine, which also inhibits the initiation of translation in a
manner indistinguishable from that of DG (17), probably acts
by its incorporation into mRNA. The antitumor activity of 8azaguanine is most likely related to its ability to inhibit protein
synthesis. However, DG was different from 8-azaguanine in
that DG had a specific effect on DNA synthesis as well as
protein synthesis.
Classical inhibitors of protein synthesis in eukaryotes also
decrease the rate of DNA synthesis with less of an immediate
effect on RNA synthesis. In mammalian cells, cycloheximide
does not inhibit total RNA synthesis at concentrations many
times higher than those eliciting maximal depression of protein
synthesis, and the RNA effect is delayed. The pattern of inhi
bition of DNA synthesis resembles that of protein synthesis
more closely (24). Low concentrations of emetine produce no
appreciable depression of RNA synthesis, whereas protein and
DNA formation are reduced to 10 and 20% of control values,
respectively (4). Puromycin inhibits protein synthesis more than
that of nucleic acids, and DNA synthesis appears to be more
depressed than RNA synthesis (12). Of the 3 drugs, the pattern
of inhibition of macromolecular biosynthesis produced by DG
qualitatively resembles most closely that of emetine. The exact
role of the inhibition of initiation of protein synthesis by DG in
its antitumor action or even in the concurrent inhibition of DNA
synthesis and subsequent cell death is still unresolved.
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CANCER
RESEARCH
VOL.
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42
3-Deazaguanine: Inhibition of Initiation of Translation in L1210
Cells
Rachel S. Rivest, David Irwin and H. George Mandel
Cancer Res 1982;42:4039-4044.
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