Download Ribosomes of Mouse Liver following

(CANCER RESEARCH 33, 1796-1803, July 1973]
Ribosomes of Mouse Liver following Administration of
Dimethylnitrosamine1
Gary M. Williams2 and Tore Hultin
Wenner-Gren Institute, University of Stockholm, Stockholm, Sweden \T. //.], and Department of Pathologv and Fels Research Institute, Temple
University School of Medicine, Philadelphia, Pennsylvania 19140 [G. M. W.\
SUMMARY
Administration of dimethylnitrosamine (DMNA), 50
mg/kg, to mice for 2 hr resulted in a 60% inhibition of
hepatic protein synthesis. This effect was accompanied by a
disaggregation of both free and membrane-bound polysomes. The increased monomers in the membrane-bound
fraction remained associated with the membranes. Isolated
total monomers from DMNA-treated mice were equivalent
to control monomers in their utilization of the synthetic
template polyuridylic acid. Also, isolated polysomes from
both groups had the same endogenous amino acid-incor
porating activity. The lack of a direct action of DMNA on
the state of aggregation of polysomes was demonstrated by
the finding that cycloheximide, which arrests the readout of
polysomes, largely protected the polysomes from disaggre
gation by DMNA.
damage is apparent from the finding that microsomal RNA
(41) and rRNA (22, 28) is highly methylated by DMNA.
Nevertheless, the functional activity of the generated mon
omers has not been tested.
This paper reports that isolated ribosome monomers
from DMNA-treated mouse liver are normal as regards
their activity in translating a synthetic messenger. Further
more, the ribosomes that are membrane bound remain
attached after disaggregation to monomers. Isolated poly
somes were also found to be functionally normal following
treatment, and the possibility of a direct destruction of
mRNA on polysomes was further rendered unlikely by the
finding that arrest of polysome readout by cycloheximide
partially protected against the effect of DMNA.
MATERIALS
AND METHODS
Materials
INTRODUCTION
DMNA3 will induce liver tumors in a variety of species
and, as with many other hepatocarcinogens, toxic doses
cause a marked inhibition of hepatic protein synthesis (18).
Although a number of disturbances in the protein synthetic
apparatus have been found following DMNA treatment, the
primary critical lesion has not been identified (19). Adminis
tration of DMNA results in a rapid disorganization of the
endoplasmic reticulum (3, 9, 11, 26) and a disaggregation
of polysomes (25), which parallels the disruption of protein
synthesis (41). The in vitro amino acid-incorporating activ
ity of microsomes isolated from treated liver is depressed (8,
12) and unfractionated ribosomes (25) and microsomes (20)
have been found to be more responsive to stimulation with
synthetic messenger. These latter results were interpreted as
reflecting a loss of mRNA from polysomes due to attack on
the mRNA (20, 25). However, the potential for ribosomal
1This work was supported by the research grants from the Swedish
Cancer Society and the Swedish Natural Science Research Council.
2The work was performed during the tenure of a Research Training
Fellowship awarded by the International Agency for Research on Cancer.
3The abbreviations used are: DMNA, dimethylnitrosamine; poly U,
polyuridylic acid; HM, homogenizing medium; LM, light medium; TCA,
trichloroacetic acid; PMS, postmitochondrial supernatant; DOC, sodium
deoxycholate.
Received February 28, 1973; accepted April 16, 1973.
1796
DMNA was obtained from Eastman Kodak Co., Roches
ter, N. Y. It was purified by distillation and dissolved in
0.9% NaCl solution on the day of use. The sources of other
chemicals were: cycloheximide, Sigma Chemical Co., St.
Louis, Mo.; poly U, Miles Chemical Co., Kankakee, 111.;
Soluene 100, Packard Instrument Co., Downers Grove, 111.;
DL-leucine-I4C (34 Ci/mole), DL-phenylalanine-14C (48 Ci/
mole), and 14C-labeled orotic acid (61 Ci/mole), Radiochemical Centre, Amersham, England.
Buffer A: 25 mM KC1:5 mM MgCl2:35 mM Tris-HCl
(pH 7.7 at 25°).Buffer B: 50 mM KC1:5 mM MgCl2:35
mM Tris-HCl. HM: 0.25 M sucrose in Buffer A. LM: 0.15
Msucrose in Buffer A.
Animals
Female white mice of the Naval Medical Institute strain,
weighing 25 to 30 g, were fasted overnight before treatment.
In the early morning, experimental animals were given i.p.
injections of DMNA, 50 mg/kg body weight, and controls
were treated with an equal volume of 0.9% NaCl solution.
Sacrifice was by decapitation and the gallbladders were
removed before the livers were extirpated and chilled in LM.
Except in the study of in vivo leucine incorporation, the
livers of 5 animals were pooled for each experimental point.
CANCER RESEARCH
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VOL. 33
DMNA-treated
Mouse Liver Ribosomes
sedimented through layers of 1.38 M and 2.0 M sucrose,
while membranous material is trapped at the interface of the
sucrose layers. It has been shown (31) and confirmed in this
work (Table 4) that following the 1st centrifugation for
sedimentation of free ribosomes some free monomers
remain in the overlying 2.0 M sucrose cushion. Therefore,
this cushion was removed, diluted, and layered over a new
cushion of 2.0 Msucrose in Buffer A placed on the pellet of
free ribosomes. The tube was oriented in the centrifuge with
the pellet outward and centrifuged for 20 hr at increased
centrifugal force (50,000 rpm, Spinco rotor 50 Ti). The
membrane-bound ribosomes were liberated by homogenization in 1% DOC and centrifuged along with the free
ribosomes through a 2.0 Msucrose cushion.
The efficiency of the separation was tested in the
following way, using isolated, l4C-labeled free monomers as
markers. Mice were given i.p. injections of 4 ¿tCi14Clabeled orotic acid. After 2 days, the food was removed, and
they were fasted for an additional 2 days to increase the
monomer content in the liver. Monomers were collected and
pelleted as described. The pellet was suspended in LM and
Preparation and Fractionation of Ribosomes
added as part of the medium used in the homogenization of
normal liver. Free and membrane-bound ribosomes were
Total Ribosomes. Minced livers were homogenized in 2.5
separated according to the procedure for obtaining sedimen
volumes of HM in a glass homogenizer fitted with a tation profiles. After the initial 24-hr centrifugation, the
motor-drived Teflon pestle. The PMS was prepared by
distribution of radioactivity in the gradient was determined
centrifuging the homogenate for 7 min at 15,000 x g. It was by pipetting alternate 0.1-ml samples of the gradient as well
added to one-ninth volume of a 10% DOC solution and
as aliquots of the PMS and suspended pellet onto filters.
gently mixed on ice. In a modification of the method of The filters were placed in ice-cold 10% TCA; washed twice
Munro et al. (27), the ribosomes were sedimented through a
5.0-ml cushion of 1.0 M sucrose in Buffer A by centrifuga- in cold 5% TCA; and dehydrated in cold and then warm
(2:1:1), and
tion for 2.5 hr at 50,000 rpm in a Spinco 50 Ti rotor at 4°. absolute ethanol, ethanol:ether:chloroform
ether. Radioactivity was counted as above.
The A260:A280 ratio of the ribosomes was always around
1.4.
Monomers. DOC-treated PMS, 1.7 ml, was applied to a Sedimentation Analysis
35-ml 15 to 40% sucrose gradient in Buffer A. This was
centrifuged for 4 hr at 27,000 rpm in a Spinco SW27 rotor.
Samples were suspended in Buffer A containing 5%
The gradient was monitored by displacement with heavy mouse liver cell sap and applied to a 5-ml 15 to 55% linear
sucrose solution through a Beckman Model DK-2 recording sucrose gradient in Buffer A. Gradients were centrifuged in
spectrophotometer provided with a LKB 4712A-4 flow cell, a Spinco SW 50.1 rotor for 65 min at 47,500 rpm (4°).
and the monomer peak was collected. The monomer Sedimentation profiles were recorded at 260 nm as de
fraction was layered on 3.5 ml of 1.5 Msucrose Buffer B and scribed.
centrifuged for 16 hr at 40,000 rpm in a Spinco 40 rotor.
The A260:A280ratio was constantly around 1.4.
Polysomes. The sample and gradient were identical to Isolation of Free and Total Ribosomes
that used for monosomes, but the gradient was centrifuged
Isolation was performed by the method of Blobel and
for only 2 hr. The lighter portion of the polysome region was
composed mainly of smaller aggregates with low amino Potter (5) in which minced livers were homogenized in H M
acid-incorporating activity due, at least in part, to a with 15 strokes to produce full liberation of free ribosomes.
substantial content of monomers. Therefore, only the These were isolated from the PMS by sedimentation
medium and heavy fractions, which had comparable activi through layers of 0.5 M and 2.0 M sucrose in Buffer A, and
ties, as Wettstein et al. (45) have found, were studied. These total ribosomes were pelleted in the same manner from
fractions were sedimented as for the monomers. The PMS made to 1% DOC.
A2io:A280 ratio was always about 1.4.
In vivo Incorporation of Leucine
Mice were given i.p. injections of 8 /¿CiDi.-leucine-14C
per 100 g body weight and were sacrificed 15 min later.
Livers were homogenized in 30 volumes of LM and replicate
1.0-ml aliquots were made to 10% TCA; Celile, 10 mg/ml;
and nonradioactive leucine, 1.5 mg/ml. After heating at 90°
for 30 min, the precipitated protein was collected by
filtration on Munktell 20/150 paper discs overlayered with
10 mg Celite. The precipitates were washed with 15 ml 5%
TCA and dehydrated by successive 10-ml washes with
2-propanol, 2-propanol: ether (1:1), and ether. The filters
were placed in counting vials, and the precipitates were
allowed to dissolve in 0.5 ml Soluene for 2 hr. Counting was
performed in a Packard scintillation counter at 55% effi
ciency in 10 ml of scintillation fluid composed of 0.5% PRO
and 0.015% POPOP in toluene. The protein content of the
homogenate was measured by the method of Lowry et al.
(17) for the calculation of specific activity of proteins.
Replicates agreed within 10%.
RNA Analyses
Separation of Free and Membrane-bound Ribosomes for
Sedimentation Analysis
In studies in which some samples might contain DNA,
the method of Scott et al. (34) was used throughout. In the
Separation was performed by a modification of the case of ribosomes in which no other fractions were analyzed,
method of Blobel and Potter (7) in which free ribosomes are RNA was measured by a modification of the method of
JULY 1973
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1797
Gary M. Williams and Tore HuitÃ-n
Ogur and Rosen (29) as previously described (13). The
values obtained by the 2 methods were closely similar.
Table I
In vivo incorporation of leucine- "C into protein
Treatment
In Vitro Amino Acid Incorporation
cpm/mg protein
Control
(3)"DM
hr(2)DMNA,
N A, 50 mg/kg, l
50 mg/kg, 2 hr (4)697
inhibition
145*388
±
119275
±
±1344461
Cell sap was prepared by homogenizing in 2 volumes of
LM the livers of mice fasted overnight and centrifuging the
PMS for 90 min at 50,000 rpm in a Spinco 50 Ti rotor. The
" Numbers in parentheses, number of animals.
supernatant was freed of amino acids by passage through a
' Mean ±S.E.
column of Sephadex G-25 (42) and stored frozen at -20°
until use.
Table 2
The incubation mixture contained, in 0.15 ml: 20 ^1
In vitro amino acid-incorporating activity of total ribosomes
ribosome suspension in LM (15 A260 units; 10 to 20 /ig
RNA); 75 n\ cell sap (about 1.5 mg protein); 1 mM ATP;
cpm/IO/ig
0.25 mM GTP; 10 mM phosphoenolpyruvate; pyruvate
RNA°
% inhibition
kinase, 40 ¿tg/ml;90 mM KC1; 19 amino acids excluding
phenylalanine (42); and DL-phenylalanine-14C, 0.67 ¿uCi/ml.
Control
71
DMNA, 50 mg/kg, 2 hr
32
55
MgCl2 was added in concentrations ranging from 6 to 18
mM. Incubation was performed in a shaking water bath for 5
'At 7 mM MgCl2.
min at 35°and was stopped by placing the samples on ice.
Aliquots of 0.1 ml were immediately pipetted onto filter
papers and processed by the method of Mans and Novelli
(21). Radioactivity was counted as above. One incubation
500
was performed in each experiment without ribosomes
added. This blank had a constantly low isotope content that
was identical to nonincubated complete mixtures and was
subtracted from the counts of the experimental samples.
Incorporation was linear for 8 to 10 min and proportional to
400
added ribosomes up to 3 times the amount normally used.
The assay conditions were established to allow maximal
incorporation in the presence of poly U. The ratio of mg
protein cell sap to mg ribosomal RNA was greater than
100:1 (27). Poly U stimulation was maximal at a concentra
g 300
tion of 900 Mg/m'- Higher concentrations did not depress
ir
incorporation and so 1200 ng/m\ was rountinely used to
S.
assure saturation.
i
o
200
RESULTS
Effect of DMNA on Protein Synthesis. Treatment with
DMNA at a dose of 50 mg/kg produced a large depression
of in vivo hepatic leucine incorporation after 1to 2 hr (Table
1). In order to study the functional activity of ribosomes at a
point where protein synthesis was severely inhibited, we
performed the experiments reported below using a 2-hr
interval of treatment at this dosage, except where noted.
This period of treatment was felt to be sufficiently short to
consider our observations to reflect the status of the
elements studied during the evolution of inhibition.
Total ribosomes isolated from the DMNA-treated mice
had a correspondingly reduced activity of endogenous in
vitro amino acid incorporation (Table 2). Optimal incorpo
ration for both types of ribosomes was found at 6 to 8 mM
MgCl2. Similar to what others have found (20, 25), the
treated ribosomes responded to the addition of poly U with
a greater increment in incorporation than control ribosomes
(Chart 1). In addition, the Mg2+ optimum was shifted from
about 10 mM for the controls to about 11 to 12 mM for the
1798
CL
O
100
9
10
11
Mg2+ CONCENTRATION
(mM)
12
13
Chart I. In vitro poly U-stimulated polyphenylalanine synthesis of
total ribosomes from control and DMNA-treated mouse liver.
treated animals. Dialyzed cell sap or pH 5 enzymes (14)
from DMNA-treated animals yielded the same or slightly
better incorporation per mg protein as control material.
Effects of DMNA on Free and Membrane-bound
Ribosomes. DMNA is activated by a microsomal enzyme
system (19), and it might therefore be suspected that
CANCER RESEARCH
VOL. 33
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DMNA-treated
Mouse Liver Ribosomes
membrane-associated polysomes would be more severely
affected by reactive metabolites than their free counter
parts. This possibility was not substantiated by the finding
that the sedimentation profiles of both free and membranebound ribosomes contained greatly increased peaks of
monomers following DMNA treatment (Chart 2). DMNA
treatment has been reported to cause a detachment of
ribosomes from the endoplasmic reticulum (3, 9). If this
were to occur under the present conditions, then this effect
should be reflected by an increase in RNA in the free
fraction and a corresponding reduction in the content of the
membrane-bound fraction. Analysis of the RNA in these
fractions as separated for sedimentation profiles revealed no
such alteration following treatment (Table 3, Method 1).
Since the total recovery by this method seemed low, an
alternate method for isolation of free ribosomes (5) was also
tested. In this experiment, the recovery of free ribosomes as
a percentage of the PMS was slightly greater, and again
there was no increase in RNA in the free fraction after
DMNA (Table 3, Method 2).
In the 2 above methods for the separation of free
ribosomes, these particles pass through the rapidly ac
cumulating membrane fraction during sedimentation. Some
evidence has been presented in support of the view that
monomers recovered from the membrane fraction represent
essentially free particles that have not been completely
separated during centrifugation (40). Since no shift of RNA
from the membrane-bound to the free ribosome fraction
was observed during the DMNA treatment (Table 3), the
identity of the monomers in the membrane-bound fraction
was further investigated. For this purpose the distribution of
added free monomers, prelabeled with I4C-labeled orotic
acid, was studied in the gradient used for separating free and
membrane-bound ribosomes. Less than 5% of the added
radioactive monomers were retained in the membranebound fraction even when material below the interface was
included to assure recovery of all membranous material
(Table 4). This small proportion, which may include heavy
subunits, is insufficient to account for the amount of
monomers found in the membrane-bound fraction and
indicates that these particles do not represent entirely an
artifact of separation. An interesting incidental finding was
the extensive trapping of monomers in the 2 M sucrose
cushion overlying the free pellet. Because of this observation
BOUND
the procedure for separating free and membrane-bound
ribosomes was modified to give more complete sedimenta
tion of the free particles (cf. Chart 2 and Table 3). The
distribution experiment was also performed with labeled
total free ribosomes in which 7% of the counts were as
monomers. In this case, only 0.6% of the counts were
retained in the membrane-bound fraction. It was felt that, if
monomers were attached to endoplasmic membranes as it
seemed, these monomers should be released after lysis of the
membranes by DOC, thereby resulting in a larger monomer
peak than would be present in the same material not treated
with DOC (43). Therefore, an aliquot of PMS was diluted
with the same volume of LM as that of DOC used on
another aliquot. Sedimentation profiles revealed in contrast
to the finding of Webb (43) that DOC treatment resulted in
a considerable increase in the monomer peak size (Chart 3),
Chart 2. Sedimentation analysis of separated free and membrane- consistent with the release of monomers from membranes.
bound ribosomes from control and DMNA-treated mice.
The PMS from DMNA-treated mice contained a larger
peak of free monomers than the control, and the magnitude
Table 3
of the increment following DOC treatment was greater than
RNA in fractions of rat liver
that observed with control PMS. This latter result could
Table 4
inPMS
Distribution of "C-labeled free monomers on gradient after separation of
brane-bound0.620.700.58'0.81'Totalrecovered2.32C2.25e1.841.93
sepa
ongradient4.233.962.382.54Totalfree1.771.551.261.12Free
as%PMS42.039.253.044.0Totalmem
free and bound ribosomes
ration"1"yTreatmentControlDMNAControlDMNATotal
Methodof
total cpm
inPMS1004.844.243.194.0
A.B.C.D.E.F.G.Total
gradientAbove
in PMS placed on
sucroselayersBetween
interface of 2 M
" Average of 2 experiments of each kind.
* Analyses performed on fractions obtained during separation for
sedimentation profiles (cf. Chart 2).
"Sum of free plus membrane bound.
" Isolation of free and total ribosomes in PMS according to the method
of Blobel and Potter (5).
' Calculated as difference between total recovered and free.
JULY
visibleband
interface and last
ml)Totalof material (0.8
fraction(B
in membrane-bound
C)Remainder
+
sucroseFree of 2 M
pelletTotal
recovered (D + E + F)cpm534016888256246123005017%
1973
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1799
Gary M. Williams and Tore Hull in
80S
TOP
BOTTOM
Chart 3. Size of 80 S monomer peak in control (A) and DMNAtreated (B) PMS with and without DOC treatment. In the samples not
treated with DOC, the absorbance curve in the polysome region indicates
light scattering due to the presence of membranes.
somes (16, 44) and the polysome profile remains fixed (10).
This effect has been used to probe the susceptibility of such
arrested polysomes to direct attack by other inhibitors of
protein synthesis (1, 10, 31, 37, 38). The mouse is less
sensitive than the rat to cycloheximide (38), but administra
tion of 100 mg/kg body weight i.p. for 1 hr followed by a
2nd injection of 25 mg/kg resulted in an 86% inhibition of in
vivo incorporation of leucine. The incomplete inhibition of
protein synthesis was reflected by an increase in the
monomer peak after cycloheximide alone (Chart 5, cf.
Chart 2). Under these conditions, when DMNA, 50 mg/kg,
was given with the 2nd dose of cycloheximide, 25 mg/kg, for
1 hr there was a definite reduction in the amount of
polysomes disaggregated to monomers by DMNA (Chart
5). In the Sprague-Dawley rat, cycloheximide, 2 mg/kg,
given for 30 min produced a 76% inhibition in in vivo leucine
incorporation. The effect of a subsequent injection of
DMNA, 200 mg/kg, for 40 min on polysome disaggregation was markedly diminished (Chart 5). In both the mouse
600
reflect a larger component of monomers attached to
membranes after DMNA treatment and strengthens the
<
interpretation that monomers are indeed bound to mem
tr
branes. Fractions corresponding to the monomer peaks
400
were collected, and the A320was obtained to determine the
content of ferritin (27). This was minor and did not differ for O
any of the monomer peaks. Therefore, the A26owas not OÕT>
00
corrected.
Functional Activity of Isolated Monomers and Polysomes.
Isolated monomer fractions from livers of control and
DMNA-treated mice were only slightly contaminated by o
ce
heavy subunits and dimers, as indicated by the sedimenta
200
tion profiles (not illustrated). They had very little or no
endogenous amino acid-incorporating activity. Poly U was CL
utilized equally well by both types of monomers as a O
template for the synthesis of polyphenylalanine (Chart 4),
and the stimulated activity of the monomers was greater
than that of total ribosomes (cf. Chart 1). The Mg2+
dependence of the reaction was the same for both types of
monomers with the optimum incorporation occurring at a
Mg2+ concentration of 11 to 12 mM.
1214
16
10
18
The phenylalanine-incorporating activity of isolated polyMg2+ CONCENTRATION (mM)
somes was measured over a MgCl2 concentration range of 6
Chart 4. ¡nvitro poly U-stimulated polyphenylalanine synthesis of
to 12 mM. Optimum endogenous incorporation for both
isolated monomers from control (O) and DMNA-treated (•)mouse liver.
control and DMNA polysomes occurred at 9 to 10 mM
Mg2+. The peak activity of the DMNA polysomes did not
Table 5
differ from that of controls (Table 5). The addition of poly
In vitro amino acid-incorporating activity of isolated polysomes at ¡0mM
U did not result in very much increase in incorporation
i and average of valuesfor medium and heavy fractions of polysomes
apparently because of the absence of monomers for utiliza
tion of the template. Consequently, the Mg2+ optimum was
cpm/lOfigRNA
not raised in the presence of poly U.
Effect of DMNA after Cycloheximide Treatment. Cypoly
U95
cloheximide reduces the rate of polysome readout (44)
Control
through inhibition of elongation (2, 15, 16). This results in
DMNAEndogenous54
51+
86
an equilibrium shift toward a high concentration of poly
1800
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DMNA-treated
MOUSE
Chart 5. Sedimentation analysis of mouse and rat ribosomes after
treatment with DMNA, cycloheximide (cyclo) plus DMNA, and cycloheximide.
and the rat, cycloheximide produced a shift to heavier
polysomes. This is regularly observed with cycloheximide
(15).
DISCUSSION
In the above experiments the interval of DMNA treat
ment was kept short in the hope that alterations giving rise
to the inhibition of protein synthesis could be detected. It
was found that disaggregation of polysomes occurred
without detachment of the monomers from endoplasmic
membranes, as determined in 2 different ways. In DMNAtreated animals, the association of ribosomes with endoplas
mic membranes has been previously studied only with the
electron microscope. Some observers have not described a
decrease in the ribosome content of the endoplasmic
reticulum (26) while others have observed degranulation (3,
9). The latter studies (3, 9) were performed after longer
intervals of treatment than the present and it may be that
degranulation is a subsequent event to disaggregation.
There is considerable evidence that monomers remain
adherent to membranes after in vitro runoff (6) and
treatment with a variety of agents that result in polysome
disaggregation (4, 6, 32, 33). Thus, the present results are in
agreement with these other observations, indicating that
there is a binding of monomers to endoplasmic membranes
that is not dependent upon polysome integrity.
The inhibition of protein synthesis by DMNA was
quantitatively reproduced in vitro by total ribosomes from
treated animals. Previous reports that the cell sap factors
involved in protein synthesis are normal after DMNA (8,
12, 20) were confirmed. The reduced activity of treated
ribosomes was accompanied by an increased responsiveness
to stimulation with poly U as others have described (20, 25).
In addition, we further documented that the maximal
stimulated incorporation with poly U occurred at a higher
Mouse Liver Ribosomes
Mg2+ concentration for the DMNA-treated ribosomes (11
to 12 mM) than for control ribosomes (9 to 10 mM). In view
of the fact that both control and DMNA-treated isolated
monomers had a Mg2+ optimum of 11 to 12 mM for poly
U-stimulated incorporation, we believe that the shift in
Mg2+ optimum of DMNA-treated total ribosomes may be
due to more effective utilization of poly U at the higher
Mg2+ concentration by the greater proportion of monomers
present. A similar increase in the Mg2+ optimum for poly
U-stimulated incorporation has been previously reported
for ribosomes from livers of animals exposed to hepatotoxins (36) and the interpretation was offered that this effect
may be due to an altered capacity of the treated ribosomes
to bind bivalent cations (35, 36). The monomers generated
under these conditions have not been directly tested as in the
present study. Vernie et al. (39) have recently shown that
membrane-bound but not free polyribosomes from DMNAtreated liver have an altered Mg2+ plus Ca2+ dependence for
endogenous incorporation. The significance of this effect
has not yet been determined.
The isolated monomers from DMNA-treated livers were
functionally as active in utilizing poly U as control mono
mers. Bearing in mind that utilization of a synthetic tem
plate is not entirely analogous to translation of an endog
enous messenger, it is concluded that the generated mono
mers are normal as regards this function. This finding com
plements the observations that the stability of that of
rRNA is not decreased after methylation in vivo by
DMNA (22) and that DMNA-generated monomers and
those run off during in vitro protein synthesis are similar
in their sedimentation characteristics in low Mg2+ concen
tration (40).
Isolated polysomes were contaminated by monomers to a
very slight degree as reflected by the small increment in
incorporation in the presence of poly U. This indicates that
the 16 hr of centrifugation did not lead to extensive runoff of
monomers. The polysomes from DMNA-treated livers
repeatedly showed activity comparable to controls, which is
in contrast to the finding of Mizrahi and DeVries (25) that
treated polysomes are less active. This reduced activity was
related by them to polysome instability (25). The same
laboratory has subsequently suggested from less direct
studies that reduced activity might be due to smaller
aggregate size of polysomes from DMNA-treated liver (40).
This seems unlikely from our result, in agreement with
Wettstein et al. (45), that the in vitro activity of aggregates
is constant for fractions from which monomers have been
excluded. Our finding indicates, therefore, no impairment of
the activity of the ribosomes on engaged mRN A at the time
when protein synthesis was markedly inhibited. This conclu
sion is strengthened by the further finding of a partial
protection by cycloheximide on the DMNA-induced break
down of polysomes. This observation is similar to that
recently reported by Stewart (37) and implies that the effect
of DMNA does not occur entirely through a direct attack
on the connecting mRN A strand. The duration of cyclohex
imide inhibition of protein synthesis was too short to
attribute its protective effect to a reduction of DMNA
metabolism and indeed Stewart (37) has shown that there is
JULY 1973
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1801
Gary M. Williams and Tore HuitÃ-n
Liver Cells after Dimethylnitrosamine, 2-Fluorenamine, or Prednisono change even after longer intervals of cycloheximide
lone
Treatment Studied by Electron Microscopy. J. Nail. Cancer
treatment. The lack of complete protection is undoubtedly
Inst., 30: 1045 1075, 1963.
due, at least in part, to the fact that in vivo protein synthesis
12. HuitÃ-n,T., Arrhenius, E., Low, H., and Magee, P. N. Toxic Liver
was not entirely inhibited by cycloheximide. These results
Injury. Inhibition by Dimethylnitrosamine of Incorporation of La
with polysomes do not support the earlier suggestions of a
belled Amino Acids into Proteins of Rat-Liver Preparations in Vitro.
DMNA-induced lesion in polysomal mRNA (20, 24, 25).
Biochem. J. 76: 109 116, 1960.
Since the above experiments revealed no defect in the 13. HuitÃ-n,T., Naslund, P. H., and Nilsson, M. O. Dissociation of
translational capacity of DMNA-treated monomers or
Ribosomes from Dormant Cysts of Anemia salina in Potassium-free
polysomes, we are led to suggest that the critical lesion may
Media. Exptl. Cell Res., 55: 269 274, 1969.
be in the process of initiation of protein synthesis. A 14. Keller, E. B., and Zamecnik, P. C. The Effect of Guanosine
Diphosphate and Triphosphate on the Incorporation of Labelled
possibility not precluded by the present studies of isolated
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monomers is a defect of these particles in reacting with the
normal initiation codon, AUG. Furthermore, since the 15. Kisilevsky, R. The Regulatory Parameter of Protein Synthesis Most
Affected by Ethionine and Cycloheximide. A Comparison of Compu
utilization of poly U as a template at high Mg2+ concentra
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tion does not require the ribosome-associated protein
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synthesis initiation factors (23, 30), another possibility is
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that these factors could be impaired or deficient. On the
Ribonucleic Acid. Biochemistry, 10: 2348-2356, 1971.
other hand, evidence has been found that DMNA methy- 17. Lowry. O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.
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Protein Measurements with the Folin Phenol Reagent. J. Biol. Chem.,
193: 265 275, 1951.
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The present findings of no direct effect of DMNA on 18. Magee, P. H. Toxic Liver Injury. Inhibition of Protein Synthesis in
Rat Liver by Dimethylnitrosamine in Vivo. Biochem. J., 71: 606 611,
engaged mRNA indicate that the defect in mRNA would
1958.
have to be an impairment of stable or newly synthesized
19. Magee, P. N., and Swann, P. F. Nitroso Compounds. Brit. Med. Bull.
mRNA to initiate new peptide chains.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the excellent technical assistance of
Birgit Lundberg.
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1803
Ribosomes of Mouse Liver following Administration of
Dimethylnitrosamine
Gary M. Williams and Tore Hultin
Cancer Res 1973;33:1796-1803.
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