[CANCER RESEARCH 30, 515—527,February 1970]
The Dependence of DNA and RNA Synthesis on Protein Synthesis
in Asparaginase4reated Lymphoma Cells
K. A. 0. ElIem, AngelinaM. Fabrizio,and L. Jackson
Departments of Pathology and Medicine, Jefferson Medical College,Philadelphia,Pennsylvania19107
SUMMARY
L-asparaginase
(EC 3 . 5 . 1 . 1 . ) (7, 8). Mutant
6C3HED tumor which were not dependent
We studied
the effects
of asparagine depletion
on macro
molecular synthesis in an asparagine-dependent line of the
6C3HED lymphoma maintained in suspension culture . Aspar
agine was removed from the culture by the asparaginase
present in guinea pig serum or by deleting it in formulating
the medium. Protein, RNA, and DNA synthesis were
measured
by the incorporation
of labeled precursors
and the
relative rates of synthesis of the individual types of RNA
were estimated by a dual-label technique which involves
chromatographic
fractionation. Guinea pig serum caused a
reproducible
sequence
of inhibitions
synthesis.
Protein
synthesis
declined
exponential
followed
kinetics.
by a more
An
initial
gradual
falling
of macromolecular
first with biphasic,
precipitous
decline
off. Accompanying
was
the
second phase was an exponential decline in DNA synthesis
with a similar half-life. Later RNA synthesis fell in the
definite temporal and quantitative sequence of rRNA >
DNA-like RNA > tRNA. Both the transcription and the
maturation of rRNA were inhibited. Dual-label autoradi
orgaphy showed that the inhibition of DNA synthesis was not
caused by blocking of the initiation of S-phase but by
slowing the rate of DNA synthesis in each cell with a
resultant prolongation of S-phase. Omission of asparagine
from the medium resulted in a similar sequence of inhibition
of macromolecular
synthesis but of more gradual onset and
progression.
Cycloheximide,
a specific inhibitor of protein
synthesis, rapidly produced
a similar sequence of inhibition
of nucleic acid synthesis. The effects of asparaginase
on
nucleic acid synthesis
thus seem to be secondary
to the
@
inhibition of protein synthesis.
INTRODUCTION
It is now recognized that certain transplantable
tumors are
lines of the
on exogenous
asparagine could be selected by their ability to replicate in
vitro in the absence of the amino acid (8). These aspar
aginase-resistant lines (6C3HED Asn@) have high levels of
asparagine synthetase (34, 37), an enzyme- which is inducible
under conditions of asparagine depletion. Recently , studies
have been conducted on the effects of GPS2 (45) and of
asparagine deletion (10) on macromolecular synthesis in
6C3HED Asn—cells. Both studies showed a rapid decrease in
protein synthesis. A subsequent decrease in precursor
incorporation
into
RNA and DNA were observed
by Sobin
and Kidd (45), who noted the analogy between this and the
cessation
of DNA
synthesis
after
the inhibition
of protein
synthesis in other systems.
Because deprivation of a required amino acid has revealed
the presence of important regulatory mechanisms for nucleic
acid synthesis in auxotrophic bacteria (29), we have explored
the effects of L-asparagine deprivation on nucleic acid
metabolism in the 6C3HED lymphoma cell in the hope of
observing some similar mechanisms and simultaneously
exploring the derangement
in macromolecular
synthesis
caused by L-asparaginase in the sensitive line of lymphoma
cells.
MATERIALS
AND METHODS
CUltUre of 6C3HED Lymphoma Cells. Cultures of 6C3HED
lymphoma cells were established from the ascitic form of the
Gardner lymphosarcoma, which was carried in C3H mice by
intraperitoneal injection of 0.4 ml ascites fluid obtained after
1 week of growth. The cells used for culture were obtained
after the injection of 0.5 ml sterile 0.02% Versene in 0.9%
NaCl solution into the ascites to prevent clotting on harves
ting. The cells were then centrifuged out, and the ascites
plasma was removed. The cell pellet was resuspended in
auxotrophic
for asparagine . This followed from the observa
tions that normal guinea pig serum had dramatic antitumor
activity in vivo foi@the Gardner lymphosarcoma
Asn— lymphoma) (26), and that the effective
(6C3HED
agent was
‘Thiswork was supported by USPHS Grants CA-05402-09 and
GM-00813.07, and an Institutional Small Grant GRS 1967—07
through a General Research Support Grant FR-54i4.
Received February 3, 1969; accepted July 23, 1969.
2GPS, guinea pig serum; Asn,
asp@agine dependence@ MAK,
methylated albumin-Kieselguhx; Q1-RNA, ribosomal precursor RNA
as separated by MAK chromatography; Q2RNA, DNA-like RNA
eluted by salt from MAK; TD-RNA, tenaciously bound (not eluted
from MAK by 1.6 M NaG solution) DNA-like RNA; D-RNA,
DNA-like RNA; L-i RNA, incompletely characterized RNA eluting
from MAK between tRNA and DNA as explained in text; 5 S, 18 S,
and 28 S - RNA, conventional sedimentation coefficients defining
ribosomal
RNA's; SDS, sodium dodecyl
sulfate..
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515
K. A. 0. Ellem, A. M. Fabrizio, and L. Jackson
growth medium at a concentration of 1.5 X 106 cells/mI.
The cells grew readily in suspension in McCoy's 5A spinner
medium (Grand Island Biological Company) supplemented
with amino acids at 25% of the concentration present in
Eagle's modified medium (15) and 25% fetal calf serum. The
cell concentration was maintained between 2 and 6 X 106
cells/mI and the cells were doubled in 25 to 35 hours. The
cultures were fed daily by replacing approximately half the
culture volume with fresh medium to reduce the cell concen
tration. The cell line has remained susceptible to the
inhibitory effects of GPS asparaginase. Because this line
demonstrates
an asparagine
dependence,
in conformity
Cell viability
was estimated
by eosin exclusion.
All cell counts were performed with hemocytometers.
GPS was obtained from Dr. Helga Suld who ascertained
that its asparaginase level was 2.61 i.u./ml before and 2.55
i.u./ml after heat inactivation of nonspecific factors (in
which form it was used) for 30 mm at 56°. The method of
assay has been previously described (48). Cycloheximide was
obtained from Nutritional Biochemicals Corp., Cleveland,
Ohio.
Measurement of Rates of Synthesis of Macromolecules.
Experiments
were
of two basic
types.
First,
for the deter
mination of the rate of total protein and total ribonucleic
acid synthesis or total nucleic acid synthesis the incorpora
tion (during a prescribed interval) of uniformly labeled
leucine-' 4C
(Nuclear-Chicago,
Des
Plains, Ill.
6.6
mCi/mmole), uridine-5-3 H or cytidine-5-3 H (Nuclear-Chicago,
1 to 5 Ci/mmole), respectively, at 37°into the acid-insoluble
components of a given number of cells was used. Where the
temporal sequence of changes in these parameters was to be
determined
2 suspended
cell cultures, the control and the
experimental,
were set up in spinner flasks. Duplicate 1-ml
aliquots were taken from the suspensions
at the required
periods and incubated at 37°in stoppered lO-ml Erlenmeyer
flasks with the 2 labeled precursors, with thaking for the
requisite interval. At the end of the incubation the flasks
were chilled in ice, diluted with 5 ml ice-cold balanced saline
and the cells filter collected on cellulose acetate membranes
(pore size, S /1). The residue in the flasks was rinsed onto
the membranes with another 5-ml aliquot of the cold
balanced saline. The membranes were washed free of acid@
soluble materials with two 10-mi volumes ice-cold 5%
trichloroacetic
acid
vial, and digested
solubilizer
to
in succession,
overnight
render
the
placed
in a scintillation
isotopes
into
and processing it in the same fashion as the
supporting the cells. Cell-associated counts were
more
than
medium
from
100 times greater
a
sample
a
toluene-soluble
form. Radioactivity was determined in a
liquid scintillation counter after the addition of a scintillation
mixture
(Spectrafluor,
Nuclear-Chicago)
as previously
through
another
than these background
values,
which were, however, duly subtracted.
Second, for the determination
of the relative rates of
synthesis of the individual nucleic acid species between 2
different
cultures
the recently
described
dual-label
method
(18) was used. Briefly, the 2 cultures to be compared (e.g.,
asparagine
depleted
and control)
were divided
into 2 aliquots
of 5 ml each (2 to 5 X 106 cells/mI) Unlabeled cytidine (to
of 10 j.@g/ml) was added to all aliquots
and then to one of each pair cytidine-5-3 H (5 Ci/mmole to 2
j.zCi/ml and to the other cytidine-' 4C (6.8 mCi/mmole to 0.5
pCi/ml) was added. The aliquots were placed in a metabolic
shaker at 37° for 40 mm. At the end of this time
suspensions
were chilled and the cells were removed
the
by
centrifugation (1000 X g, 3 mm). The cell pellets were lysed
into the usual solution of sodium dodecyl sulfate, and the
1 4C-labeled
control
was
mixed
with
the
3H-labeled
experi
mental lysate (Set I) and vice versa (Set II). The 2 mixed
lysates were then deproteinized,
the nucleic acids were
precipitated with ethanol, washed, and analyzed by methyl
ated albumin-Kieselguhr chromatography
as detailed else
where (18). The experimental results are expressed as the
rate of synthesis of a particular nucleic acid species in the
treated (e.g. , asparagine depleted) cells as a percentage of its
rate in the control cells. The calculations depend only on the
3H/' 4C ratios of the peak fractions of the separated nucleic
acids and provide a value analogous to a geometric mean of
the 2 separately analyzed groups (Sets I and II). The values
were calculated with the formula
100 X
The
method
eliminates
@J[(3H/'4C)IJX
minimizes
any procedural
[(‘4C/3H)II].
column-to-column
differences
variation
and
in handling the speci
mens which might otherwise influence the analysis of the
nucleic acids from each group. It also cancels out any bias the
cells have in handling the 2 different isotopes.
When the cell concentration differed between the 2 groups
(as in Tables 3 and 4) the relative rates were corrected for
this difference by multiplying the relative rates by: viable
cell concentration of control sample/viable cell concentration
of treated
sample (cf
Ref. 18).
The variation in the method stems from 2 sources: random
counting errors and cell counting and sampling errors. The
first leads to an error which is expressed
with 0.5 ml Nuclear-Chicago
incorporated
the
membrane
membrane
a final concentration
with
genetic terminology, it will be referred to as 6C3HED Aso
to distinguish it from the asparaginase-resistant, asparagine
independent
variants which occur (6C3HED Asn@) and
which have been isolated from this line. (A. M. Fabrizio, in
preparation).
refiltering
in the tables as the
95% confidence interval about the least certain value, which
was for the nucleic acid sample which provided the fewest
counted disthtegrations@ The formula used for calculating the
95% confidence interval of the product [(3H/' 4C)IJ X
[(‘4C/3H)II] was
+ (l/C2)1
described ( 18). Quench correction
and cross-contamination
of isotopes was determined
with the channel ratios method
@
a /@[(iiH,)+(l/@@@(l/H2)
+ (1/C2)]
with an external standard; polynomial curve fitting to the
standards and the calculations were performed with an IBM where H,, C1, H2, C2 are the means of the counts recorded
360/30 computer.
for the duplicate aliquots for H and ‘C in the 2 sets of
Blank values for the membranes were determined by columns. Because the relative rates of synthesis are the
516
CANCER RESEARCH VOL 30
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Asparaginase Inhibition of Macromolecular Synthesis
square root of the product the 95% confidence interval is
closely approximated by (a/2) X 100% (39). In the cases
where a tube-by-tube comparison of rRNA was made the 95%
confidence interval was calculated with
a
2'sJ?i/hi)+(l/c,
+ (1/h2) + (I/c2)
where h1, h2, c,, c2, are the actual number of disin
tegrations counted in the samples from the 2 sets of
tubes (i.e., not means of duplicates as for the other
@
@
in other nucleic acid moieties (17) and dpmt is the total
amount of isotope in Tube t. d@ is the ratio of D-RNA in a
tube of corresponding position, relative to the A260 profile,
to the TD-RNA of the actinomycin D-treated sample in
Chart 1. The values for Pt are listed in Table 1 and these
are similar
to values
calculated
for HeLa cells. The relative
rates of isotopic incorporation into rRNA for each tube were
then corrected for the contaminating D-RNA by using the
easily derived formula R,. (R@p@X RTD)/l —Pt where R@
is the uncorrected
relative
rate of RNA synthesis
in Tube
t
nucleic acid fractions). It represents a maximal variation for
evaluating the significance of difference between the values
and RTD @5
the relative rate of TD-RNA synthesis.
Autoradiography:
After exposure of the cells to labeled
for the individual nucleic acid species within an experiment.
The second source of variation leads to a factor of uncer
tainty which affects all the values to the same extent and
produces a 95% confidence interval of approximately 10%
for the overall difference between treated and control group.
For assessment of the rate of maturation of rRNA in
relation to the rate of transcription of rRNA, we made use
of the fact that MAK chromatography crudely separates the
acetic acid (3 : 1), and air dried on glass slides. The slides
were then coated with NTB-3 emulsion. exposed, developed,
and stained with Giemsa. When 3H and ‘
4C were both
present in a specimen the slides were further treated by
coating the first developed emulsion with celloidin to filter
out ‘
H disintegrations and developed. This method shows
species
in the first layer (2).
of rRNA,
which
elute
in the order
18 S. 18 S, and
28 S and high molecular weight precursors (Q, -RNA) ( I 7).
The resolution is poor, but by analysis of each successive
tube and determination
of the relative rate of isotope
incorporation into the broad classes of mature and precursor
RNA, an approximate estimation of the rate of maturation can
be made. Tubes from the elution of Set I and Set II
nucleic acids resulting from the dual-label design (see above),
which corresponded to one another on the basis of their
position in relation to the absorbance marker profile of the
rRNA, were matched. The relative rate of synthesis of RNA
in that region of the rRNA elution was calculated from the
@ii/'4C ratios of the matching tubes, yielding a value Rt for
Tube t. With the short labeling periods used in this study the
DNA-like RNA which elutes with salt contributed a signifi
cant amount of the isotope present in the rRNA zone.
Because D-RNA was less responsive to the changes being
studied the contamination reduced the real magnitude of the
alteration in rates of synthesis of rRNA, As can be seen in
Chart 1 some isotope was Incorporated into RNA in the
sample treated with actinomycin D (which suppressed rRNA
synthesis), which elutod with the absorbance peak of rRNA,
in front of the Q2 .RNA peak. It has previously been shown
that this material resembles D-RNA in composition rather
than rRNA (12). The actual amount of isotope in D-RNA in
each tube across the rRNA zone of the control cell elutlon
can be calculated if the assumption is made that actinomycin
D treatment does not alter the relative amounts of D.RNA
eluting as Q3 .RNA and tenaciously bound D-RNA. In this
regard there is no significant difference in the relative
amounts of Q2.RNA and TD-RNA from HeLa cells labeled
In the presence of the same concentration of actinomycin D
for periods ranging from 20 mm to 2 hr (K, A. O@Ellem and
S, L. Rhode, unpublished results). The formula giving the
proportion (ji@,)of Isotope present in D-RNA In a Tube t in
the rRNA zone of an elutlon Is: Pt
d1 X dpmTD/dpmt
where dpmTD Is the amount of isotope In the TD-RNA
fraction which is used as a measure of the total D.RNA
fraction because It is relatively uncontaminated with isotope
precursor
14 C
labeling
they
in
were
the
centrifuged,
second
emulsion
washed,
layer
fixed in ethanol:
and
H
plus
“
C
RESULTS
Effects of GPS on Total Macromolecular Synthesis. Sobin
and Kidd (45) studied the effects of normal GPS on
macromolecular synthesis of 6C3HED Asn cells growing in
vivo in the ascites form. To confirm their overall conclusions
we have studied the effects of GPS on 6C3HED Asn cells
growing in vitro in greater detail because of the greater
reproducibility of the in vitro system. The overall rates of
protein, RNA, and DNA synthesis were estimated by
measuring the amount of leucine-1 4C, uridine-5-3H, and
thymidine-3 H, respectively, incorporated into aliquots of a
suspension culture during a definite interval of incubation
after various periods of time had elapsed after the addition
of GPS to the culture. In Chart 2A a comparison of the
effects of GPS on protein and RNA synthesis over a 16-hr
period is illustrated. Two aliquots were taken from a culture
4 hr after routine medium replenishment. To one of them
GPS was added to a final concentration of 2%. At intervals,
thereafter, duplicate one 1-mi aliquots were removed from
each and the rates of protein and RNA synthesis measured
by the simultaneous Incorporation of leucine-' 4C (0,5 i.@Ci/ml)
and uridlne.3H (1 pCi/mi) for 1 hr. The acid-soluble
isotope was removed and the cells were solubilized and
counted as in “Materials and Methods,― The rate of
incorporation into the GPS-treated cells is expressed as a
percentage of the control cell rate. The dramatic fall in
leucine-' 4C Incorporation into protein within the first 2 hr
after adding the GPS was characteristically biphasic The
inhibition of RNA synthesis was more gradual and was not
obviously biphasic. Because the effects of adding GPS was
precipitous a more detailed study was made of macro
molecular synthesis in the first 2 hr of GPS action. Charts
2B and 2C show a comparison of DNA, RNA, and protein
synthesis in cells taken from a culture 24 hr after refeeding
in the presence and in the absence of 2% GPS. Protein
synthesis was most rapidly affected by GPS so that Its rate
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517
K. A. 0. Ellem, A. M Fabrizio, and L. Jackson
TUBENUMBER
Chart. 1. MAK elution profiles of a mixture of nucleic acids from 6C3HED lymphoma cells labeled for 40 mm with cytidine-3H (control cells) and
cytidine-'4C (cells exposed to actinomycin D 0.03 j@Ci/mlshowing selective inhibition of isotope incorporation into Q1-RNA and rRNA. For
simplicity the tRNA and the DNA eluted from this column are omitted. The portions of the chromatogram shown are the ribosomal RNA complex
(Tubes 30 to 40), and the elution of TD-RNA with a convex gradient of guanidine thiocyanate (from 0 to 6M, at 35°)followed by a wash with 6M
P nidinethiocyanateat 80 (tubes 65 to 90) (8). The absorbancesacrossthe ribosomalpeak, @--@;3H dpm, o---o, ‘4C
dpm, .- - -..The ratio
4C/311
is plotted
as o—o,
illustrates
the selective
inhibition
of rRNA,
and reveals
the presence
of D-RNA
denoted
as Q2-RNA.
second, more gradual phase had an exponential decline with
a half-life of 84 min [difference was highly significant (p <
0.01)] . The DNA synthesis decayed with a half-life of 86
mm in the first 1.5 hr. It is perhaps significant that the
synthesis of DNA and protein have similar decay rates
(determined by linear regression) after 20 mm when they
were both declining together [there was no significant
difference between them (p > 0.9)] . The decay of RNA
synthesis was somewhat slower than that of DNA synthesis
and from Chart 14 it can be seen to have a half-life
approximating 2 hr.
The Effects of Om@on of Asparagjne from the Medium on
Gross Macromolecular Synthesis. For comparison of the
effects of deletion of an exogenous supply of asparagine
with those due to the GPS L-asparaginase similar experi
ments were performed to the above after resuspending Asn
cells in either McCoy's medium (control) or McCoy's
medium without L-asparagine. The results of a typical
Chart 2D shows a semiogarithmic
plot of the data expressed
experiment are shown in Chart 3, which demonstrates that
as a percentage of the control rates of incorporation of although there was a decline in protein synthesis and RNA
leucine-' 4C and thymidine-3H,
respectively.
The chart synthesis after the deletion of asparagine, the decline was
clearly shows that the rapid decline of protein synthesis more gradual than that produced by treatment with GPS,
preceded the decline in DNA synthesis by 15 to 20 mm. The the half-life of synthesis of protein being about 9 hr and
l4hr.
decay of protein synthesis again was biphasic with an initial thatofRNAbeing
There was a decline in RNA synthesis succeeding the fall in
exponential decline (to about 40% of the control rate) which
lasted for 20 mm and had a half-life of 11.1 mm. The protein synthesis after GPS treatment and after deletion of
fell to less than 40% of the control rate within 40 mm of
adding the GPS. Chart 28 shows that in this same time
interval the effects on total RNA synthesis led to a small
degree of inhibition, whereas Chart 2C shows that the
synthesis of DNA was profoundly affected. Although the
effects of GPS on protein synthesis .were immediate there
seemed to be a lag of about 30 min before the precipitous
decline in DNA synthesis began. Precursor incorporation into
protein, RNA, and DNA of the control cells showed a small,
gradual fall over the 2 hr of the experiment. Because the
culture was used without refeeding it , this decline can be
attributed to the effect of depletion of the nutrients, an
effect which wifi be detailed elsewhere.
Because the inhibition of protein synthesis seems to begin
immediately while the decline in DNA synthesis seems to be
delayed 20 to 30 mm after adding the GPS, a more precise
analysis of the kinetics of the inhibitions was made by
taking samples at 5-mm intervals after the addition of GPS.
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CANCER RESEARCH VOL.30
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Asparaginase Inhibition of Macromolecular Syn thesis
Q2-RNA was often not distinctly separated from Q, -RNA
Table 1
for periods of labeling longer than 30 mm ( 18). This was
especially noticeable with the lymphoma nucleic acids. For
confirmation
that a Q2-RNA moiety was present but
obscured in the rRNA complex the selective and virtually
asparagine361217tRNA95•3a67.465.554.1tRNA+5SRNA98.364.658.744.6L-1
deletion of
complete inhibition of rRNA (and Q, -RNA) synthesis by
NucleicacidHoursafter
low doses of actinomycin D was used (12, 18). Accordingly,
aliquots of a suspension of cells were labeled in the presence
or absence of actinomycin D (0.03 @.tg/ml)with cytidine-' 4C
Inhibition ofnucleic acid synthesis by omission of asparagine
from the medium of 6C3HED cells
or cytidine-3H,
respectively,
30 mm after adding the actino
RNAb97.063.549.714.7DNAC71.448.7(47.2)9.54.9rRNA99.144.829.723.7Q1-RNA100.050.733.124.6D-RNA94.663.341.3323Total
mycin D. After 40 min incubation the cells were centrifuged
(1000 X g, 3 mm) and lysed in SDS. The 2 lysates were
mixed and deproteinized; the nucleic acids were applied to a
MAK column.
acids94.455.933.324.495%
nucleic
confidence intervals±2.1±1.9±2.7±2.8
aThe values represent the rate of synthesis of the nucleic acid fraction
as a percentage
of the control
rate, during the 40 mm after the elapsed
hours of asparagine depletion. The determinations were made by the
dual-labeltechnique and MAKchromatography as described in the text
bL..i RNA possibly represents 5 S RNA.
CThe DNA values were calculated from the amounts of isotope
incorporated into the DNA peak as it elutes from the MAKcolumn. A
small amount of the isotope (approximately 10%) is present in RNA,
and this tends to reduce the magnitude of the change in DNA synthesis
when DNA is the primary target of the effects under study. The effect
is small as can be seen from the value in parentheses which was
calculated from the alkali-resistant isotope present in the DNA peak
dAs described in “Materials
and Methods―the 95% confidence
interval was calculated for the samples with the least number of
recorded counts. This figure defines the maximal interval of uncertainty
at this level of significance for comparison of rates between different
nucleic acids within an experiment.
asparagine from the medium. This also occurs in bacteria
starved for an amino acid for which they are auxotrophic
[e.g., Maaloe and Kjeldgaard (29)] and is selective for rRNA
in them (33). Therefore, we next examined the rate of
synthesis of the individual nucleic acid types for any signs of
selectivity of the asparagine deprivation on them.
MAK Analysis of Lymphoma Nucleic Acids. The method
used for analyzing the synthesis of the nucleic acids of
6C3HED cells was MAK chromatography.
The elution
pattern of the nucleic acids was found to be similar to those
of other
mammalian
cells and, in particular,
to that obtained
by Lingrel (28) with rabbit bone marrow cells. The order of
elution by a gradient from 0.3 to 1.6 M NaCl-0.05 M
tris-HC1, pH 6.7, was tRNA, tRNA and 5 S RNA, Ll -RNA,
1 -RNA,
and
Q2
-RNA,
(e.g.,
Refs.
1 7
,
39).
Ll
-RNA
has
not
been fully characterized but is rapidly labeled, is detected in
both nucleus and in polysomes, and has a guanine 1- cytosine
composition and sedimentation resembling 5 5 RNA (39).
It may represent some of the 5 5 RNA of the cells. The bulk
(60 to 70%) of the D-RNA was retained tenaciously by the
column, but it could be eluted by raising the pH of the
eluting fluid, or by adding hot 1.6 M NaCl solution or 2%
SDS (e.g., Ref. 17). Recently, it has been found that a
concentration gradient of guanidine thiocyanate elutes the
TD-RNA with some selectivity because it can fractionate the
TD-RNA from different sources (19). The RNA types that
eluted in the rRNA region were poorly resolved so that
Chart 1 shows the pattern of rRNA and Q2 -RNA eluted by
the NaCI gradient and of TD-RNA eluted by a 0 to 6 M
guanidine thiocyanate gradient. The salient features were the
gross inhibition of incorporation of cytidine .@4C (actinomy
mycin D-treated cells) into the ribosomal RNA area, with
relative sparing of RNA which eluted in a position corre
sponding to Q2 -RNA (1 2, 18). There was a small amount of
residual labeling of the RNA eluting with the A260 peak of
rRNA (Tubes 31 to 34) which was less than the equivalent
of 25% of the amount of isotope present in the same tubes
from the control cells (cytidine-3 H). This is mainly con
tributed by D-RNA (12). Thus, after a short incubation with
labeled precursor, when relatively little of the isotope in
rRNA has been processed from precursor rRNA (Q, -RNA)
to mature 18 S and 28 5 rRNA, a significant amount of the
isotope in the tubes containing the 18 5 and 28 5 RNA was
present as D-RNA. The values for this contamination (Table
2) represent upper limits and possibly overestimate it
depending on the presence of residual rRNA synthesis. There
was no selective inhibition of labeling across the guanidine
thiocyanate elution pattern of TD-RNA except for a barely
significant inhibition in the first peak (Tube 66) most easily
seen in the fall in the
‘4C/3H ratio in that tube,
and this
can be attributed to inhibition of the small amount of
isotope in the contaminating rRNA which elutes in that peak
(19). As with other cells in culture (12, 18) this dose of
actinomycin D has a 10-fold difference in its effect on the
transcription of rRNA and D-RNA. Thus the D-RNA of
6C3HED cells is fractionated into Q2 -RNA (about 40% of
the D-RNA) and TD-RNA which constitutes the remaining
60% in a fashion similar to that of other cells.
The method used for quantitation of the rates of nucleic
acid synthesis was the dual-label technique recently described
(18) (see “Materialsand Methods―). Because the two samples
being compared by cochromatography may be recognized by
the different isotopes in them the influence of asparagine
depletion on Q2 -RNA can be partially assessed despite the
absence of a discriminable Q2-peak. Because Q1 -RNA is a
precursor to rRNA the rate of incorporation of labeled
precursor into
1-RNA is the best measure of the trans
cription rate of rRNA from the rDNA cistrons, while the
rate of appearance of label in mature rRNA is further
influenced by the intermediate steps involved in the conver
sion (reviewed
in Ref.
14). Any differences
noted
the rates for rRNA and Q1 -RNA incorporation
between
may thus
FEBRUARY 1970
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519
K. A. 0. Ellem,A. M Fabrizio,
and L.Jackson
2B
70
2oo
0
8
U
$50
0
I,
i40
0
C
0
0
30
a
7
0
6
@0 5
C
2
@0
0
C
20
40
60
Minuts@ Aft.r
80
-100
120
Adding GPS
•0
‘0
E
C
•0
10(
Minut•sAft.r
5
Adding GPS
15
25
35
Minutes Aft.,
45
55
Adding GPS
65
75
Chart 2. Kinetics of inhibition of protein, RNA, and DNA synthesis after addition of GPS (2%) to cultures of 6C3HED Asn cells. The synthesis
of protein, RNA, and DNA were measured by the incorporation ofleucine-' 4C, uridine-5-3H, and thymidine-5-3H, respectively, as described in the
text. Protein and nucleic acid synthesis were measured simultaneously in the same samples by taking advantage of the 2 different isotopes. Each
point is the mean of duplicate aliquots. A, decline of RNAand protein synthesis over a 16-hr interval after the addition of GPS to a 6C3HEDAsn
culture. Synthesis in the GPS-treated culture is expressed as a percentage of the control value. Labeling time was 1 hr. B and C, more detailed
comparison of the decline in RNA and DNA synthesis in relation to protein synthesis in the 2 hr after GPS treatment. The control and GPS-treated
cells were with the precursors at the concentrations described in the text, for 10 mm at the indicated time intervals. Bars, range of duplicate
@
aliquots. Control values are plotted as open symbols; GPS-treatedvaluesare solid symbols. n—o,protein synthesis; o—o, RNA synthesis;
DNA synthesis. D, detailed kinetics of GPS inhibition of DNAand protein synthesis. Cellswere labeled for 10-mmperiods; aliquots were taken for
labeling at 5-mm intervals after addition of GPS to culture. Exponential decline of DNA synthesis (o—o) and biphasic exponential decline of
protein synthesis (.—.) are revealedby the semilogarithmicplot.
indicate alterations
its precursor.
Pattern
@inthe rate of maturation
of Inhibition
of Nucleic
of rRNA from
Acid Synthesis
Resulting
from Asparagine Deficiency. The gradual decline in RNA
synthesis caused by deletion of asparagine from the medium
was analyzed for evidences of selectivity.
Two appropriately sized aliquots were taken from a culture
520
and centrifuged (200 X g for 10 ruin). The cells were
resuspended in McCoy's medium lacking asparagine. The
control
sample was made 0.34 mM with asparagine
while the
other was left deficient in asparagine. Both samples were
then incubated in spinner flasks and aliquots were removed
after different intervals of time for exposure to labeled
cytidine for measurement of the rates of nucleic acid
CANCER RESEARCH VOL.30
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Asparaginase Inhibition of Macromolecular Synthesis
DNA-like RNA were also severely inhibited. The incorpora
tion into tRNA and 5 5 RNA was less inhibited.
The values calculated
for rRNA synthesis were different
when rates based on the isotope
chromatographing
S to 28 5 RNA area were compared
in the 18
with those of the
Q, -RNA peak. Therefore, a tube-by-tube comparison of the
rates of rRNA synthesis was made across the zone of elution
of the rRNA complex to obtain an estimate of the matura
tion of rRNA, as explained in “Materialsand Methods.―
Table 2 embodies these results. The upper set of figures
which have not been corrected for D-RNA contamination
show a tendency, in the more severely inhibited cultures, for
the matured RNA (18 5 and 28 S rRNA) to be more
inhibited
@
@
than the precursor
rRNA (Q, -RNA). The lower set
of figures has been corrected for D-RNA contamination on
the basis previously described and these show that the
maturation
process was more markedly inhibited than the
Hours After Deleting Asparogine
transcription of the ribosomal precursor RNA (Q,) by a
Chart 3. Declinein RNA and protein synthesis in 6C3HEDAm- cells factor of ‘
/@ to /@ at the later time periods.
resuspended in McCoy's medium lacking in asparagine. The incorpora
Pattern of Inhibition of Nucleic Acid Synthesis Induced by
tion was measured and expressed as in Chart 2, control cells being GPS. The rates of synthesis of the individual nucleic acids
resuspended in McCoy's medium containing asparagine. u—u, protein
were determined in the 6C3HED cells after GPS had been
synthesis; .—., RNA synthesis.
added to the medium, relative to the same cells in normal
medium. Two aliquots were taken from the same culture 4
hr after refeeding to stabilize the stimulatory effect of fresh
synthesis by the dual-label technique described in “Materials medium (K. A. 0. Ellem, A. M. Fabrizio, and L. Jackson, in
and Methods.― Each aliquot was divided in 2 and cytidine
preparation),
so that
the cells would
be in a similar
was added to 10 j.zg/ml and cytidine-3H (2 pCi/ml) or nutritional status to those used in the preceding experiments
4 C (0.5
@.tCi/ml) was added
to 1 of each pair. After
on asparagine deletion. To one of them GPS was added to
40 mm isotope incorporation the cells were collected by a final concentration of 2%; the other was left as a control.
centrifugation and the H-labeled cell pellet of 1 pair added
After incubation for different time intervals after adding the
to the ‘
4C-labeled sample of the other, each mixture being GPS, aliquots were taken, labeled with cytidine-3H or -‘
4C as
lysed with SDS and deproteinized with phenol. The mixed described in the previous section and analyzed by the
samples (each containing a treated and control lysate) were dual-label MAK method. The results in Table 3 (Columns 1
processed and fractionated on MAK. The relative rates of and 2) show a similar sequence of inhibition of nucleic acid
incorporation
were calculated
from the
H/' 4C ratios of synthesis to that resulting from asparagine deletion tabu
either pooled fractions, or from individual tubes in the lated in Table 2. The speed of the shutdown is, however,
rRNA-Q2 -RNA area of the elution, as described in “Materials very much faster. Thus, in the interval 10 to 50 miii after
and Methods.―
the GPS was added DNA synthesis fell by 60% and rRNA
The values in Table 1 represent the percentage rate of synthesis fell by 24%. After 2 hr of GPS action the pattern
synthesis of the individual nucleic acids of Asncells relative of inhibition of the nucleic acids resembled that of cells
to the control cell rate. It can be seen that 3 hr after which had been deprived of asparagine for 17 hr (Table 2,
deletion of asparagine from the cells there was no significant Column 4). The same selectivity of inhibition, viz., DNA >
depression of RNA synthesis in any of the RNA types. rRNA > D-RNA > tRNA, was observed in both sequences.
Pattern of Inhibition of Nucleic Acid Synthesis after
However, most significantly, there was already detectable an
inhibition of precursor incorporation into DNA. By 6 hr Inhibition of Protein Synthesis by Cycloheximide. The fall in
(Column 2) the inhibition of DNA synthesis was much more nucleic acid synthesis always followed a decline in protein
marked (53%) and every RNA species now showed depressed
synthesis induced by the asparagineless state. For the
examination of the hypothesis that the nucleic acid events
incorporation.
The inhibition
of RNA synthesis was not
uniform but was selectively marked on ribosomal RNA. The could be simply secondary to the interruption of protein
synthesis, the pattern of nucleic acid inhibition after drug
extent of inhibition of the transcription
of ribosomal
mediated termination of protein synthesis was explored.
precursor
Qi -RNA was 5 1%. The low-molecular-weight
RNA's (tRNA, 5 5 RNA, and Ll -RNA) were inhibited to Aliquots of cells from a culture refed 4 hr previously were
handled in a similar fashion to the previous experiments
the same extent as the DNA-like RNA of the cell, all about
except that instead of adding GPS, cycloheximide was added
35%.
group in a final concentration of 10 @zg/ml.
With increase of the time of incubation of the cells in the to the “treated―
absence of asparagine, the inhibition of incorporation of Columns 3 and 4 of Table 3 record the relative rates of
synthesis of the individual nucleic acids in the intervals 10 to
isotope into DNA became greater than 90% of the control
rate (by 12 hi) while the synthesis of ribosomal and 50 and 60 to 100 mm after the addition of the cyclo
FEBRUARY 1970
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521
x: A. 0. Ellem, A. M Fabrizio, and L Jackson
Table2
Effects on the maturation ofrRNA in 6C3HED Asncells
ofgrowth in the absence of asparagine
A260
of3
@b
mediumRNAmarker―
1217ValuesSetI
Rtc18
Seth
Hours elapsed after deletion of asparagine fromthe
3
6
calculatedfrom totalacid-insoluble3H/14Cratios in each tube
29.723.7A260
S + 28 S RNA
0.140
0.140
99.1
50.5
32.224.149.0Q,-RNA
Peak
0.220
0.185
98.8
44.8
0.160
33.124.60.095
0.120
100.0
50.7
34.027.10.070
0.100
35.027.70.045
0.060
34.725.60.035
0.040
99.4
98.2
99.1
54.2
56.0
57.1
98.6
946±1.7
58.2
63.3±1.2
35.324.9TD@RNA=RTDd
0.035
1.995%
confidence
intervalValues
[email protected]
8.17.7A260Peak
28.520.8Q1-RNA
32.223.7Factor
41.3±2.132.3±
con'ected for D-RNA contamination
0.6510
0.2868
0.1005
107.5
100.5
100.6
26.5
37.4
49.2
maturationdecrease
of
gR18S+28S/RQ,
1.07
0.54
0.250.36
a@f@e@indicates the predominant form of ribosomal RNA present in the tube. The predominant
speciespresent in the A260 peak is 28 S rRNA with much 18 S rRNA also present.
bFor illustration of the method of tube matching in the ribosomal RNA elution zone the A260 readings
for the 2 sets of tubes fromthe ribosomalRNA areaof the elution from the 2 dual-labeledanalysesare
given for the first time period. Wheze any difficulties in matching arose from phase differences between
sets valuesw@ecalculated for each of the 2 fits and the geometric means of these valueswere used.
CRt is the relative rate of synthesis of RNA in each tube of the rRNA complex calculated by using the
total isotope incorporated into each area.
dRTh is the relative rate of synthesis of TD-RNA.
CRr is the corrected value for rRNA uth@gthis formula which allows for D-RNA contamination: R,. =
(Rt-p RTD)/1 —p.
‘pis the fraction of total RNA of control cells present as D-RNA in each tube. The values were
calculated as described in the text.
(These values do not take into account the possible effects which decrease in the maturation rate may
have on the apparent rate of transcription due to delay in processingthe precursors.
heximide. The patterns of shutdown in RNA synthesis were
similar
to those
after
the addition
of GPS, and again DNA
synthesis was most dramatically
affected. The major
difference was in the immediacy of the inhibition with the
cycloheximide, which can be correlated with the almost
instantaneous
@
@
effectiveness
with
which
it shuts
off protein
synthesis (13, 20, 56).
The rate of the maturation of rRNA from its precursor to
the mature 18 S and 28 5 RNA forms was again assessed by
a tube-by-tube analysis of the rRNA complex eluted from
MAK. Table 4 compares the results for GPS and cyclo
heximide. In both cases the processing of the precursor
Q1-RNA to stable 18 5 and 28 5 rRNA components was
rapidly and almost completely inhibited when the transcrip
tion process had been reduced only by /2 and /@ There was
522
thus clear evidence of a marked dependence of the successful
maturation of rRNA on concomitant protein synthesis.
Inhibition of protein synthesis by asparagine depletion or
cycloheximide
was not accompanied
by any localized
changes in the guanidine thiocyanate elution pattern of the
TD-RNA. The inhibition of D-RNA synthesis was thus
general.
Effects of GPS on S-Phase. Because the culture consists of
unsynchronized cells it is possible that the effect of GPS on
DNA synthesis was accomplished either by preventing new
cells from entering S-phase or by decreasing the rate of DNA
synthesis in all cells in S-phase or possibly both. In order to
resolve this question the phenomenon was studied with a
two-emulsion autoradiograph technique (2). A culture was
exposed to thymidine-' 4C for 60 mm 14 hr after
CANCER RESEARCH VOL 30
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Asparaginase inhibition
Table 3
Inhibition ofnucleic acid synthesis after exposure of 6C3HED
cells to guineapig serum and cycloheximide
The times listed are the periods for which the agent (guinea pig serum
or cycloheximide) was present in the cell suspensionbefore the isotope
@
Labeling began. The period of precursor incorporation was 40 ruin. All
valuesand symbols are the same as those used in Table 1.
pigserum
(2%)Cycloheximide(10i@g/m1)10
___________________
of Macromolecular Syn thesis
tion with the serum. Control cells were similarly labeled but
were not treated with GPS. Autoradiographs were prepared
and scored as described previously. As can be seen in Tables
5 A and B the percentage of cells synthesizing DNA before
and after GPS treatment, as well as the percentage of treated
as compared to control cells in S-phase, remained essentially
unaltered. On the other hand grain counts of the H layer
show
that the rate of incorporation
of thymidine
decreased
with GPS treatment, as compared with the control cells, so
Nucleic
that in this experiment the inhibition of DNA synthesis was
acidGuinea
hr10
hrtRNA93.738.8108.469.9tRNA+5SRNA88.536.1107.166.4L-1
mm2
mm1
65% in the interval from 30 to 120 mm. This indicates that
the rate of DNA synthesis rather than the percentage of cells
in S-period was affected by the asparaginase in the serum.
RNA90.038.193.463.2DNA41.916.015.6(5.5)13.0rRNA60.722.050.236.0Q1-RNA76.028.256.038.2TD-RNA80.335.975.757.6Total
The cells used in this experiment were taken from a culture
which had been refed 14 hr previously, and were then
resuspended in fresh medium. The replenished supply of
nutrients
nucleic acids
72.0
±2.042.3
±2.529.4 ±3.361.0
95% confidence interval
±3.6
Table 4
Analysis ofthe maturation ofrRNA after inhibition ofRNA
synthesis
by inhibition ofprotein synthesis with guinaz pig serum and
cycloheximidea
(2%)
j.@g/mi10
10 mmserum
120 mmCycloheximide10
miii60
Values calculatedfrom total 3H/'4Cratios
18 S and + 28 S RNA60.7
22.0A260
[email protected]
74.2Q1-RNA76.0
76.6
34.8TD-RNA
Q1-RNA
@
of grains
from a culture
which had not been refed in the previous
24
of nutrient
depletion.
mm
in each tube (Rd
50 2
36 0
the time
40:9
36:2
examination of the distribution of the 2 isotopes among the
small population of cells which were labeled with only 1
28.2
27.9
56 0
38 2
isotope
60:1
39.5
of GPS on the initiation of DNA synthesis (3H label only) and
on the termination
77.7
30.7
65 .7
44.3
79.3
32.2
81.4
79.6
33.1
64.4
47.9
interval
of the experiment
was determined
because
75.7 ±1.4
was small (90 mm) an
it would
reflect
the effects
of S-phase (â€4̃C label only). The lower set
@g-@
initiation
and
termination.
Inasteady
state
condi
of
values
represents
the
distribution
tion a slight preponderance
57.6 ±2.3
expected
because
the
of
cells
between
of ‘
4C-labeled cells would be
cells
were
labeled
longer
with
24.2
3.9
2.6
—4.3
thymidine-' 4C than with thymidine-3H. At all of the 3
periods examined the GPS-treated sample had a relative
paucity of the cells labeled with H alone. This may imply
either a reduction in the number of cells initiating DNA
synthesis or possibly undue sensitivity of the DNA synthesis
53.8
75.5
22.4
27.3
26.9
27.6
occurring
53.8
36.0
difficult to discriminate between the light 3H- and ‘
4C-labeled
Factorof
maturation
cells and
0.32
0.05
0.14
0
aValues and symbols as for Table 2.
the
the
end
of
cells labeled
S-phase
with
which
4C alone.
would
make
Further,
it
more
DISCUSSION
replenishing the medium, and then the culture was divided in
half, and the cells were removed
at
detailed autoradiographic study will be needed to determine
which explanation is correct.
inhibition
R18S+28S/RQ,
number
The results in Tables 5 A and B show that the decrease of
DNA synthesis in the GPS-treated cultures was not due to
large shifts in the population of cells in S-phase . Although
Valuescorrectedfor D-RNA contamination (Rn
18 S and 28 S RNA
A260 P@
in the total
26.3
2.095% = RTD80.3
±1.0 35 .9 ±
confidenceinterval
@
an increase
hr. The grain counts shown in Table 6 demonstrate the
progressive decline in DNA synthesis after GPS and the slight
decline in the control cells which can be attributed to the
effects
Elution
markerGuinea
caused
over the nuclei of controls and GPS-treated cells from 30 to
90 min as had been found previously (K. A. 0. Ellem, A. M.
Fabrizio, and L. Jackson in preparation). The experiment was
repeated with just the thymidine-3H to label the cells at
defined times after adding GPS to 1 of a pair of aliquots
by centrifugation.
Both cell
pellets were resuspended in fresh medium. GPS was added to
1 aliquot , and samples were removed
and then exposed
to
thymidine-3 H for 30 mm after 30, 60, and 90 mm incuba
Production
of the asparagineless state in cultures of
6C3HED Asn— lymphosarcoma
cells is followed by a
definite sequence of inhibitions of macromolecular synthesis.
Protein synthesis declines first. This is closely followed by a
fall in the rate of DNA synthesis. RNA synthesis is then
depressed and follows a reproducible sequence temporally
FEBRUARY 1970
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523
x:
@i.a Ellem, A. M Fabrizio, and L Jackson
Table SA
Effects of GPS on thymidine incorporation into 6C3HED Asn
Cells
cellsUnlabeled3H
isotopesin 100
Time after
grains/lO
cells―Distributionof
adding GPS
(min)3H
labels30Control
@
alone‘
‘C
aloneBoth
7160Control
Serum416
15420
219
48
463
Serum740
6490Control
25225
296
68
161
Serum840
30822
283
45
270
aThe grains in the first emulsion (3H and
66
4C) were counted 2ver 20 cells by two
observers. They were then corrected for the grains caused by â€C̃ by counting and
subtracting the grains in the 3H layer caused by 14C in 20 cells in parallel
preparations, which had only been labeled with thymidine-'4C. The increase in
number of grains with time of incubation after the thymidine-' 4C labeling was due
to the refeeding effects mentioned in the text.
Table SB
counted)―Time(min)3H‘4CH/'4C30Control
Distribution
ofcelIs with only one isotope present(100 cells
0.2560Control Serum30
2070
800.43
0.2890Control Serum45
2255
780.82
Serum41
1154
890.69
0.12
@
a@e hundred cells were counted which were labeled with either H
or 4C, but not both, according to the method outlined in the text.
@
@
Four separate counts of 100 cells each on the same slide gave standard
deviation of ±2 cells for the apportioned values for 3H or C, which
was assumed to be the inherent error for the other counts, all done by
the same observer.
Table 6
Inhibition ofDNA synthesis by guinea pig serum in cells in
nutrient depleted medium
cells―ControlGPSinhibition
of grains/lO
Time after
adding GPS
(%)3019413629.96018412134.2901789049.41201687853.6
(min)No.
amino acid in adequate amounts owing to a deficiency in
asparagine synthetase, an enzyme which is present and
inducible in strains of the lymphoma which are independent
of asparagine (Asn@) (34, 37). Removal of the external
supply is followed by rapid depletion of the cell pool of
asparagine (9), and the consequent low level of asparaginyl
tRNA presumably becomes rate limiting for peptide synthesis
when an asparagine group is needed to continue the oblig
atory sequence of amino acid insertion into the polypeptides
being synthesized (41). The asparagine limitation inhibits the
assembly of asparagine rich proteins more than those with
little of this amino acid (41).
The present observation that the inhibition of amino acid
incorporation
mammalian
aCells were labeled for 30 min with thymidine-3H (1 MCi/mI)after the
indicated time of exposure of the treated aliquot to guinea pig serum.
Autoradiograms were made and the number of grains in 50 cells were
counted and averaged to a 10-cell unit.
and quantitatively
of rRNA > D-RNA > low-molecular
weight RNA's > tRNA.
Mechanism
of Inhibition
of Protein
Synthesis.
The interrup
tion of protein synthesis by removal of a supply of aspar
agine from 6C3HED Asn—cells is well documented (44, 45).
It is caused by the inability of this tumor to synthesize the
524
into
protein
is resolved
into
an initial
rapid
exponential decline followed by a more gradual one is
unexplained. The initial phase could perhaps be caused by a
pool expansion of leucine in the cell, although Broome (9)
has found evidence of a large increase in the pool size of
only aspartic acid in a similar system.
The decline in nucleic acid synthesis was found to be
similar in sequence differing only in rapidity, whether the
interruption of protein synthesis was caused by deletion of
asparagine or inhibition by cycloheximide. The differences in
speed can be correlated with the fact that cycloheximide
causes almost instantaneous cessation of protein synthesis in
cells at the concentration
used here ( 13 , 20 , 56),
whereas nutritional deprivation leads to a very slow decline
(half-life 9 hr) (possibly due to a small contribution of
asparagine from the fetal calf serum), while the fall of
protein synthesis caused by GPS is intermediate between
these (half-life of the first 60% loss was 11.1 miii; subsequent
decay had a half-life of 84 mm).
The gross anabolic derangements which result from GPS
asparaginase seem to stem from the inhibition of protein
synthesis. They do not specify the mode of cell death of
Asn— cells, but an explanation of this could perhaps be
sought in any of several metabolic systems. It is useful,
however, to consider the possible mechanisms of the effects
CANCER RESEARCH VOL. 30
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Asparaginase Inhibition ofMacromolecular
which seem to be secondary to the inhibition of protein
synthesis.
Mechanism of Inhibition of DNA Synthesis. The inhibition
of DNA synthesis by asparagine depletion like that caused
by cycloheximide
is most probably secondary to the
inhibition of protein synthesis, and this phenomenon has
been the subject of considerable investigation. Studies with
cells and organisms other than the lymphoma cells used in
this investigation also have shown that antibiotics such as
puromycin
(32, 36, 42, 52) and cycloheximide
(3, 25, 56),
which act on protein synthesis will cause a reduction in the
rate of DNA synthesis in the individual cell. In addition
more physiological means of retarding protein synthesis, such
as depletion of 2 essential amino acids in Tetrahymena
pyriformis (47), wifi also retard DNA synthesis.
The simultaneous replication of histones and DNA in
eukaryotic organisms was noted cytologically many years ago
cursor RNA into the mature species, is also inhibited. In all
these situations protein synthesis is depressed and the degree
and rapidity of onset of inhibition of RNA synthesis is
roughly correlated with that of the protein •
synthesis
although preceded by the latter. The amino acid dependence
of RNA synthesis in the 6C3HED Asn—cells as well as the
few other eukaryotic cells in which it has been studied (1 1,
53) resembles that of stringent bacteria (1 6).
That the present observations of the inhibition of pre
cursor incorporation into RNA solely reflect an increased
degradation of the newly synthesized RNA by activation of,
e.g., RNase seems improbable from the rapidity of the
effects, in the case of cycloheximide and GPS. However, an
increase in the alkaline RNase activity of the postmito
chondrial fraction of 6C3HED Asn— cells from animals
treated by injection 2 hr previously with guinea pig aspar
aginase has been reported (30). If the enzyme is only
(1, 4, 5, 38).Theconcurrence
andreciprocal
dependence
of cytoplasmic
histone and DNA synthesis has recently been demonstrated
most elegantly by biochemical methods in physiologically
synchronized cultures of HeLa cells (6, 40), and in regener
ating liver (50). Also Shah (42) used 5-methyltryptophan
to
inhibit gross protein synthesis while selectively preserving
histone synthesis. Despite a 75% fall in general protein
synthesis after 18 hr of the 5-methyltryptophan
action DNA
synthesis was unaffected. Histone synthesis was only detect
ably affected after 24 hr exposure to the analog when a 37%
depression in histone synthesis was accompanied by a
dramatic fall (80% inhibition) in DNA synthesis. This points
to histone synthesis as being a necessary and perhaps unique
concomitant of DNA synthesis.
Owing to the instability of the amide-carboxylic bond to
acid hydrolysis, most amino acid analyses of proteins do not
quote the content of asparagine (or glutamine) in them. In
Synthesis
it would
(the location which was examined in that study)
have no relevance to the inhibition of transcription
of rRNA (an intranuclear event) nor would it explain the
similar, selective effects of the 3 different initiating situa
tions studied here on the various species of RNA. It could,
however, be responsible for abortive maturation of rRNA,
for example, by leading to rapid degradation of newly
synthesized 18 S rRNA-containing subunits as they emerge
from the nuclei (e.g., Refs. 20, 46).
Studies of the effects of cycloheximide on RNA synthesis
in L-cells (20) in regenerating
rat liver (22) and in HeLa cells
(46, 54) have shown the necessity for protein synthesis in
the maintenance of a normal rate of rRNA transcription
although this dependence is not absolute (54). In these and
other studies (24, 51) with inhibitors of protein synthesis
rRNA synthesis was selectively inhibited. Further, Summers
et a!. (49) have shown that inhibition of protein synthesis
particular
we have been unable to find any published value results in the rapid disappearance of about 50% of the
for the asparagine content of any histones (also e.g., 23, assayable RNA polymerase activity of nuclei from exponen
35). Published figures for the amide content of histones vary tially growing HeLa cells but not of the residual RNA
from 2.8 to 8.1% as moles/lOO moles of all amino acids (35). polymerase of cells in stationary phase. We have observed (to
Recently Phillips (27) has been quoted as finding that 40% be published) that there is a selective depression of rRNA
of the second carboxyl groups of aspartic and glutamic acid synthesis in cells in stationary phase. There are different
residues, which are present in the various histone fractions of RNA polymerase activities for rRNA and DNA-like RNA in
calf thymus nuclei to the extent of 8 to 16%, are present in mammalian cells (3 1, 55) which may be due to different
the amide form. It is thus probable that histones contain enzymes (31). Thus inhibition of protein synthesis could
significant
amounts
of asparagine,
so that
asparagine
lead to differential loss of the rRNA polymerase if this
deficiency in Asn cells could lead to rapid inhibition of enzyme had a much shorter half-life than the extranucleolar
histone synthesis and thereby cause the inhibition of DNA enzyme. This would explain differential inhibition of rRNA
but would not explain the added decrease of
replication. It would seem significant that the rate of decay transcription
of DNA synthesis is the same as the rate of decay of protein maturation of rRNA. Alternatively, specific proteins con
synthesis after the latter has been stabilized after the pre stituting a small pool (54) may be needed for the maturation
of rRNA.
Inhibition
of protein
cipitous initial inhibition of leucine-' 4C incorporation. It process and transport
emphasizes the quantitative nature of the dependence of synthesis may lead to a critical shortage of these, which
would lead to inhibition of the maturation process, and
DNA synthesis on protein synthesis.
Mechanism of Inhibition of RNA Synthesis. Either the either by mass action or by failure to remove precursor
specific depletion of an essential amino acid or the addition rRNA from its template lead to the inhibition of rRNA
of a specific inhibitor of protein synthesis (2 1, 43) leads first transcription, which we have observed and was observed by
to a selective inhibition of rRNA transcription followed by RNA polymerase assay (49). Both of these alternative
mechanisms could be contributing to the selective inhibition
depression of D-RNA and then tRNA synthesis. Further
of rRNA synthesis. Also, in a preliminary
communication
it
more, the evidence also indicated that the maturation
was stated (53) that valine starvation of HeLa cells leads not
process, which involves the conversion
of ribosomal
pre
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525
K. A. a Ellem, A. M Fabrizio, and L Jackson
only to a reduction in the rate of transcription of rRNA
precursor but also in a slowing of its maturation to 18 S and
28 S RNA. Study of isotope incorporation and elimination
from the various intermediates
and products of rRNA
synthesis by using methods with greater resolving power in
these systems should throw further light on the regulatory
mechanisms of rRNA production and the reason for its
dependence on protein synthesis.
ACKNOWLEDGEMENTS
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527
The Dependence of DNA and RNA Synthesis on Protein
Synthesis in Asparaginase-treated Lymphoma Cells
K. A. O. Ellem, Angelina M. Fabrizio and L. Jackson
Cancer Res 1970;30:515-527.
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