- Email: [email protected]
Repression of arginase synthesis in Chang liver cells
Q 1967
by Academic
Experimental
Press
Inc.
Cell Research
48, 1-17
REPRESSION
1
(1967)
OF ARGINASE SYNTHESIS CHANG LIVER CELLS EVA
The
Wenner-Gren
Institute,
IN
ELIASSON
University
of Stockholm,
Stockholm,
Sweden
Received June 1, 1967
VARIATIONSin
arginase activity in cultured animal cells related to variations in the composition of the growth medium have been demo&rated repeatedly [3, 6, 13, 14, 20, 24, 25, 261. In an established cell strain grown in this laboratory (Chang liver) arginase activity varies at least 40-fold, from 0.05 units/mg protein [3] to 2.0 units/mg protein [6], under different growth conditions. These variations are readily reversible, and seem not to be due to a selection of cells with a different genetic constitution. Results from experiments with Chang cells also suggest that arginase activity is repressed by a product of a metabolic pathway initiated by the arginase reaction [3, 61. However, definite conclusions as to the mechanism of this regulation, which in its effect is analogous to product repression in bacteria, could not be drawn from these experiments, The aim of the present investigation was to get further information about the mechanism of regulation of arginase activity in cultured cells. Preliminary experiments with Chang cells had shown that temporary omission of a single essential amino acid from the minimal growth medium resulted in a rapid increase in the specific arginase activity of these cells, immediately after addition of the missing amino acid. This increase in enzymic activity, which was large enough to be measured within 1 hr, offered an opportunity for studying the regulation of enzyme accumulation during short time experiments. The advantages of such experiments were several: (1) The continuous changes in the environment of the cells, occurring in stationary cultures as a result of the metabolism of the cells, were less extensive during short periods of time. (2) The degradation of the enzyme was small as compared with its synthesis during the experimental time. Differences in the rate of accumulation could therefore be ascribed to differences in the rate of synthesis, rather than to differences in rate of destruction of the enzyme. (3) The effect on specific enzyme synthesis of an inhibition of DNA dependent RNA synthesis by addition of actinomycin D, required study over short periods of time, because of the general toxic effects of high concentra1 - 671819
Experimental
Cell Research
48
2
Eva Eliasson
tions of actinomycin D on cultured cells. These effects of the antibiotic were especially pronounced when the cells were grown in suspension cultures [S, 321, and resulted in an irreversible loss of viability and a rapid decrease in the overall content of protein and RNA of the cells. MATERIALS
AND
METHODS
Chang liuer cells originally obtained from Microbiological Associates, Bethesda, Maryland, were cultivated in suspension in a slightly modified Eagle medium supplemented with 10 per cent horse serum as previously reported [29]. The stock cultures, from which single experiments were started, were propagated through serial dilution with fresh medium twice a week to a cell concentration of 0.25 x lo6 cells/ml. The present experiments were started with cells from such stock cultures 48-66 hr after the last dilution. The cells, which were then in a state of rapid growth with a generation time. of 30-35 hr, were sedimented and transferred to fresh medium at a cell density of 0.5-0.7 x 106 cells/ml. In spite of the care taken to standardize the culture methods, it was impossible to avoid variations in the arginase activity of the stock cultures [6]. Comparable experiments were therefore always started with cells from the same stock culture and with serum from the same batch. Testing for contaminations were conducted as previously described [3]. No contaminations were found. The viability of the cells was determined by means of the Lissamine green permeability test [lo]. Cell counts were made either microscopically with aid of a Btirker counting chamber or in a Ljungberg celloscope 101 (AB Lars Ljungberg and Co., Stockholm). Total cell protein was determined according to Lowry el al. [16]. The cells were collected by centrifugation at 500 g for 7 min, washed with cold Hanks’ salt solution [9] and resuspended in distilled water. The determinations of total protein content were in agreement with results from the cell counts, indicating that the preparation for protein determination did not remove protein from the cells. Determination of arginase activity has been described [6] and specific enzyme activity is expressed as pmoles of urea formed per hour and mg protein. Determination of the rate of incorporation of labeled precursors into protein and RNA has also been described [5]. Samples from the cultures were incubated in media containing 0.4 mM W-L-leucine (2 mC/mmole) for IO min or 0.2 mM 14C-uridine (4 mC/mmole) for 20 min. ChemicaZs.--‘4C-labeled leucine was purchased from Radiochemical Centre, Amersham, England, 14C-2-uridine from New England Nuclear Corp., Boston, Mass., actinomycin D from Merck, Sharp and Dohme Laboratories and puromycin dihydrochloride was obtained from the Nutritional Biochemicals Corp., Cleveland 28, Ohio. RESULTS
The effect of temporary
amino
acid deficiency
on arginase
activity
Exponentially growing Chang cells were sedimented and transferred to a minimal medium from which one essential amino acid had been omitted. 0.1 mM MnCl, was included in the medium in order to stabilize arginase Experimental
Cell Research
48
Repression
of arginase
synthesis
in Chang liver cells
3
[S, 261. On the next day the missing nutrient was added to the culture at a concentration corresponding to the normal growth medium. The viability of the cells was not impaired by maintenance in a deficient medium for one day, and the normal rate of protein synthesis was restored within 2-4 hr after addition of the missing nutrient (cf. [5]). The increase in specific arginase activity after addition of glutamine or phenylalanine to cultures maintained for one day in deficient media is shown in Fig. 1. A similar increase in enzymic activity after starvation for arginine or histidine is seen in Fig. 4A (open symbols). Essentially the same effect was obtained if the prestarved cells were sedimented and transferred to a “normal” growth medium. This rapid increase in arginase activity in prestarved cells on addition of the missing nutrient occurred irrespective of which amino acid had been omitted during the starvation period, indicating that the increase of enzymic activity might be an unspecific effect of nutritional “step up” conditions after amino acid deprival. The rate of increase in arginase activity depended on the length of the starvation period as shown in Fig. 2. This experiment was conducted with parallel cultures in presence or absence of manganese and the cells were prestarved for glutamine for 0, 15 and 24 hr. The increase the enzyme
in arginase
activity-a
result of an increased
rate of synthesis
of
The immediate increase in arginase in prestarved cells restored nutritionally was prevented by the concomitant addition of puromycin (10 ,ug/ml), in-
I
10
.! 20 30 HOWlS
Fig.
1.
40
so
I
10
20
30
HOURS
Fig.
2.
Fig. l.-The effect on specific arginase activity of temporal starvation for glutamine or phenylalanine. Chang cells were incubated for 23 hr in media lacking glutamine (O-O) or phenylalanine (A-A). At 23 hr the missing amino acids were added. The growth media contained 0.1 mM MnCl,. Fig. 2.-The increase of arginase activity after glutamine addition to cells preincubated in a glutamine-free medium for 0, 15 and 24 hr. Cells were incubated in a glutamine-free medium in presence ( 00) and in absence (AA) of 0.1 mM MnCl,. At times indicated by arrows glutamine was added. Specific arginase activity 3 hr after glutamine addition denoted by filled symbols. Experimental
Cell
Research
48
Eva Eliasson dicating that synthesis of enzyme protein rather than an activation of preformed enzyme took place. If the enzyme were rapidly turning over in normally growing cells, an accumulation of newly formed enzyme molecules might be, however, the result merely of a stabilization of the enzyme protein [27]. It has been shown that arginase in HeLa cells turns over more rapidly than the average cell protein [26]. Although this seems to be true also for arginase in Chang cells the following experiment indicated that the increase in arginase at nutritional “step up” was due to an acceleration of the rate, at which the enzyme was synthesized rather than to a stabilization effect. In the experiment shown in Fig. 3 and Tables I and II duplicate cultures were started with cells from three stock cultures with different specific arginase activities. (The low activity of the stock culture from which cultures E and F in Fig. 3 were started was due to the addition of 6 mM proline to the growth medium for 10 days before the start of the experiment (cf. [6]). The cells were incubated in a glutamine-free medium for 20 hr. Glutamine was then added, and at the same time 0.1 mM MnCl,, to cultures B, D and F (open symbols).) Protein determinations indicated that the growth of the cells during the 24 hr period after the addition of glutamine was not significantly affected by the presence of manganese (Fig. 3 B). The labeling data in Table I show that the rate of ‘“C-leucine incorporation into protein at the end of the starvation period was 10 per cent of the rate of incorporation into exponentially growing cells. The rate of overall protein synthesis was, however, rapidly restored on addition of glutamine, and there was no significant difference in the rate of incorporation of 14C-leucine between the cultures to which manganese had been added and the corresponding control cultures. Differences in the increase in specific arginase activity between the cultures were therefore not due to differences in overall growth. During the first four hours after glutamine addition the rapid increase in arginase activity (Fig. 3A) occurred at about the same rate in all cultures, though manganese always had a slightly stimulatory effect. During the period 24 to 43 hr, i.e. 4 to 23 hr after glutamine addition, specific arginase activity decreased (A) or increased only slightly (E) in the absence of manganese. In presence of manganese there was a considerable increase in arginase activity during this period also, but this increase was always less rapid than during the first four hours after glutamine addition. This was true not only for the increase in specific activity but also for the increase in total activity of the I whole culture. A comparison between the development of the enzyme activity Experimental
Cell Research
48
in cultures
Repression
of arginase
synthesis
in Chang liver cells
5
B and F (grown in presence of manganese) indicates that the increase of enzymic activity in these cultures was independent of the amount of enzyme present in the cells. It seems reasonable, therefore, to conclude that destruction of the enzyme was not a determining factor for the development of the enzyme in the presence of manganese. The stability of arginase in the presence of manganese is in good agreement with earlier results from experiments with non-growing Chang cells [6] and with HeLa cells [26]. TABLE
I. Incorporation
rate of 14C-L-leucine into protein of cells from the experiment shown in Fig. 3.
For incorporation 2.5 x lo6 cells were sedimented and resuspended in 275 ~1 medium containing 0.4 m&f %-L-leucine (2 mC/mmole) and incubated for 10 min at 37°C.
Culture (Fig. 3) C
Time (hours from start) 0
Treatment Exponentially culture
20
20 hr glutamine
22
20 2 20 2
22
24 24
20 4 20 4
hr glutamine hr complete hr glutamine hr complete + MnCl, hr glutamine hr complete hr glutamine hr complete + MnCl,
of the cells
Incorp. of 14Cleucine in 10 min (CPM/w? protein)
growing stock
8000
starvation
900=
starv. medium starv. medium
7200
starv. medium starv. medium
7400
starv.
1000=
8000
7600
E
20
20 hr glutamine
E
22 22
hr glutamine starv. hr complete medium hr glutamine starv. hr complete medium + MnCl,
8500
F
20 2 20 2
E
24 24
hr glutamine starv. hr complete medium hr glutamine starv. hr complete medium + MnCl,
9400
F
20 4 20 4
cells were labeled in a glutamine-free
medium.
a Glutamine-deficient
9400
8500
Experimental
Cell Research
48
6
Eva Eliasson
In the absence of manganese the accumulation of arginase was always less rapid than in its presence. It seemed reasonable to believe that the difference in enzyme accumulation between cultures A and B, C and D or E and F respectively was due to the destruction of the enzyme in absence of manganese (camp. [26]). On th’ is assumption the destruction of enzyme, per unit enzyme present, per hour was calculated (Table II). The results indicate a destruction rate of arginase of 2 to 3 per cent/hr during the period 23 [24] to 43 hr, which is higher than the turnover rate for average cell protein in cultured cells of II. Effect of manganese on arginase accumulation in cultures started with cells with different specific activities (Fig. 3).
TABLE
Fraction Time W
Specific act.
Mean value Culture
20
of enzyme
Increase A
23
0.66
+ 0.20
-0.11
0.55 C
+ 0.25
E
0.34
= 0.02a
= 0.02
0.61 x 20 (Increase D) - (Increase (Sp. act. C) x (time) 0.31 - 0.25
F (MrPf)
0.46 x 4
C)
= 0.03a
(Increase F) - (Increase (Sp. act. E) x (time) 0.23 - 0.21
0.13 + 0.21
+ 0.23
0.24 x 4
E)
= 0.02a
0.36 0.26 - 0.07
0.36 0.38
43
D (Mn2+)
+ 0.31
Culture
0.24 0.34
0.56 x 3
0.64
0.13
24
0.24 - 0.20
0.17 t 0.11
0.33
0.58
24
+ 0.24
+ 0.17
Culture
0.33
Culture 20
A)
0.87
0.46 24
(Increase B) - (Increase (Sp. act. A) x (time)
B (Mns+)
0.70
Culture 20
Increase
of MnCl,.
0.70
0.61 43
Specific act.
0.46 0.56
0.66
in 1 hr in the absence
Culture
0.46
23
inactivated
+ 0.07
0.41
+ 0.26
0.38 x 19
= 0.03
0.62
a The difference in the increase in arginase in presence or in absence of Mn*+ during the short interval of 3 or 4 hr is near the limit of the accuracy of the determinations, but -admits a rough estimation of the rate of destruction of the enzyme. The results are in good agreement with the results of several other similar experiments. Experimental
Cell Research
48
Repression
of arginase
synthesis
7
in Chang liver cells
1 per cent/hr as determined by Eagle et al. [2]. The calculations of the destruction rate of the enzyme during the short period of rapid increase in enzyme activity during the first 3 or 4 hr after glutamine addition might be less accurate, because of the small differences between the activities attained within the short time interval, but the results indicate that the destruction of .*
HOURS
B
I 10
: 20
30 HOURS
Fig. 3.
I LO
. SO
I 10
20
30
40
50
HOURS
Fig. 4.
Fig. 3.-A, The effect of 0.1 mM MnCl, on arginase activity of cells prestarved for glutamine. Cultures A ( 0 - - - 0 ) and B ( O-o) were started with exponentially growing cells from the same stock culture. Cultures C (A - - -A) and D ( n--n) from another stock culture. Cultures E ( n - -- n ) and F ( q - q ) were started from a serially diluted culture, which had grown in presence of 6 mM proline for 10 days, and had therefore a low arginase activity. The cells were incubated in a glutamine-free medium without additions for 20 hr. Glutamine was then added and at the same time 0.1 mM MnCl, to cultures B, D and F (open symbols). B, Growth of cultures (symbols as in A); pg cell protein per ml culture. Fig. 4.-A, The effect of 6 m&f proline present in the culture medium during temporary amino acid starvation and subsequent nutritional restoration. The cells were incubated in media without arginine ( 0 - 0, w - -- w ) or histidine (AA, A---A). 23 hr thereafter the missing amino acid was added. The media contained 0.1 mM MnCl, and the media of the cultures indicated by filled symbols also 6 mM proline. B, The lack of immediate effect of 6 mM proline added together with the missing amino acid. Cells were incubated for 23 hr in a glutamine-free medium, containing 0.1 m&f MnCl,. At 23 hr glutamine (O-O) or glutamine + 6 m&f proline ( 0 -- - 0) was added.
the enzyme in the absence of manganese occurred at about the same rate during the whole experimental time. Under no conditions did the turnover rate of the enzyme appear to be high enough to account for the rapid increase in enzyme activity during the first hours after glutamine addition solely as a result of stabilization. It seems therefore reasonable to believe that the accumulation of arginase under Experimental
Cell Research
48
Eva Eliasson
8 nutritional
“step up” conditions
was due to a high rate of synthesis
of the
enzyme protein. Repression of arginase synthesis in the presence of proline The reaction
sequence to proline by way of A’-pyrroline
been suggested to be a major route for the metabolism
HOURS
Fig. 5.
5-carboxylate,
of ornithine,
has
derived
hOuRs
Fig. 6.
Fig. 5.-A. The effect on arginase activity of proline present during glutamine starvation and subsequent nutritional restoration. The cells were incubated for 21 hr in a glutamine-free medium with 6 mM proline ( 0 - - - 0 ) or without proline addition ( O- 0). At 21 hr glutamine was added. B. The effect of proline added together with glutamine to a glutamine deficient culture. Cells were incubated for 19 hr in a glutamine-free medium. At 19 hr glutamine (O-O) or glutamine + 6 mM proline was added ( 0 --- 0 ). The experiments were conducted in the absence of manganese. A continuous destruction of arginase at a rate of 2-3 per cent/hr must be taken in account when comparing the results with the results of the’ experiments of Fig. 4. Fig. B.-Effect of removal of proline at the time of glutamine addition to a culture prestarved for glutamine in presence of 6 mM proline. The cells were maintained in a glutamine-free medium in presence of 6 m&f proline for 24 hr. The cells were then spun down and transferred to a complete, minimal growth medium without proline (O-O) or to the same medium with 1 pg/ml of actinomycin D (a... n) or to a complete medium with 6 m&f proline added (O---O) (no manganese present, see legend Fig. 5).
from arginine via arginase, in mammalian cell strains [6, 291. Recent results have shown that arginase activity was repressed in Chang liver cells when proline was added to the minimal growth medium [6]. A series of experiments were conducted in order to investigate the effect of proline added to cell cultures at different times during amino acid starvation and subsequent nutritional “step up”. During the starvation period (s.f. [4]) as well as during the period later than 4 to 6 hr after nutritional “step up”, proline had always a repressing the arginase activity (Figs. 4 A and 5). Experimental
Cell Research 48
effect on
Repression
of arginase
synthesis
9
in Chang liver cells
During the first hours after the addition of the missing amino acid to prestarved cells, proline repressed arginase activity only if present already during the starvation period (Figs. 4 and 5). (If glutamine was the amino acid omitted during the starvation period the increase in arginase at “step up” was only partially repressed, while the effect of proline was more pronounced in cells prestarved for arginine or histidine.) When cells prestarved for glutamine in the presence of proline were spun down and transferred to a complete growth medium lacking proline the repression of arginase synthesis was immediately reversed (Fig. 6). Addition of 6 mM proline to the growth medium seemed to affect arginase accumulation specifically, since it had no significant effect on viability and overall growth of the cells. Nor did added proline affect the rate of incorporation of 14C-labeled leucine into cellular protein or the incorporation of 14Curidine into RNA. On the whole the results are compatible with earlier results [6] indicating that the rate of arginase synthesis in Chang cells is specifically correlated to the intracellular level of a metabolic repressor. Experiments with actinomycin gave some further information about the localization of the mechanism responsible for the regulation of arginase synthesis. A stable messenger
for arginase
synthesis
Recent results [5] have shown that a restoration of overall protein synthesis in glutamine starved Chang cells takes place within a few hours after addition of the missing amino acid. This restitution of protein synthesis occurs without a preceding increase in the prevailing low rate of RNA synthesis and is not prevented by addition of actinomycin D. It was therefore suggested that previously synthesized messenger RNA is brought into action in response to the reestablishment of an adequate cellular environment. In the two experiments shown in Table III actinomycin D (0.5 or 1 pg/ml) was added to cell cultures which had been maintained in a glutamine-free medium for 23 hr. One hour later glutamine was added. In view of the rapid inhibitory effect of actinomycin D on RNA synthesis of cultured cells [5, 21, 321 it seems reasonable to believe that RNA synthesis was effectively inhibited in the experimental cells already at the time of glutamine addition. (Four hours after the addition of glutamine the incorporation of 14C-uridine into RNA had decreased to 1 per cent of the rate in “normally” growing Chang cells.) In spite of the inhibition of RNA synthesis arginase activity increased by SO-100 per cent during a 4 hr period after the addition of glutamine. This Experimental
Cell
Research
48
10
Eva Eliasson
increase occurred at about the same rate as in the control cells in the absence of actinomycin D. These results strongly suggest that the arginase formation in “step up” cells took place in response to previously formed messenger RNA, and that this messenger had a life time exceeding 4 hr. Identical results were also attained with cells prestarved of histidine. III.
TABLE
Effect of actinomycin increase
The cells were incubated then added. Actinomycin For incorporation medium containing
Experiment I
II
D on the rate of RNA-synthesis in arginase activity.
in a glutamine-free medium (no manganese) for 24 hr. D was added 1 hr before the addition of glutamine.
of W-uridine 2.5 x lo6 cells were 0.2 mM r4C-uridine (4 mC/mmole)
Treatment
of the cells
Exponentially growing cells 24 hr glutamine starvation 24 hr glutamine starv., 4 hr complete medium 24 hr glutamine starv., 4 hr complete medium + actinomycin 24 hr glutamine starv. 24 hr glutamine starv., 4 hr complete medium 24 hr glutamine starv., 4 hr complete medi1.m i- actinomycin
and on the Glutamine
was
sedimented and resuspended in 275 and incubated for 20 min at 37°C. Incorp. of W-uridine (CPM/w? protein)
Sp. arginase act.
-
2510 740 590
0.31 0.23 0.46
1
20
0.46
-
630 690
0.37 0.70
30
0.67
Actinomycin ha/ml)
D
,ul
D
0.5 D
(If similar experiments were extended over longer periods of time a decrease in the rate of arginase accumulation occurred in the presence of these concentrations of actinomycin D. However, conclusions as to the life time of the arginase messenger could not be drawn from such experiments because of a general cell destruction, which resulted in gross morphological changes, loss of viability and a decrease in the total cellular RNA and protein.) When lower concentrations of actinomycin D (0.025-0.1 ,ug/ml) were used in similar experiments arginase accumulation during the first 5 hr after nutritional “step up” was not affected. After that time, when the increase in arginase activity in the control cultures was normally retarded, arginase activity was not decreased but on the contrary slightly stimulated in the presence of actinomycin D (Fig. 7A). Experimental
Cell Research
48
Repression Regulation messenger
of arginase
of the rate of arginase RNA
synthesis synthesis
11
in Chang liver cells on the level of translation
of a stable
In order to investigate further the effect on arginase formation of an inhibition of RNA synthesis at the time when enzyme synthesis was normally were performed. retarded in the “step up” cells a series of experiments Actinomycin D at concentrations of 0.5 or 1 ,ug/ml was added 5 hr after the addition of glutamine to prestarved cultures. The result of one of these experiments, which all gave similar results, is shown in Fig. 7 B. (The experiment was made in the absence of manganese and a continuous destruction of enzyme during the experimental time must be taken in account.) The stimulation of enzymic activity by addition of actinomycin D at concentrations high enough to inhibit most of the DNA dependent RNA synthesis, strongly suggested that the decrease in the rate of enzyme formation in the absence of actinomycin D was not due to a decrease in the amount of messenger RNA present, but rather to a decrease in the rate at which a stable messenger was translated in to enzyme protein. An alternative explanation, which cannot be excluded on the basis of these experiments, could be an inhibition of messenger break down in presence of actinomycin D (camp. [l, 311). However, further support for the hypothesis postulating a regulation of the rate of arginase formation at the level of translation of a stable messenger RNA was attained from the experiments, shown in Fig. 6. When glutamine was added to a cell culture prestarved of glutamine for 24 hr in presence of 6 mM proline the increase in arginase was comparatively slow. A stimulation of arginase activity occurred if the prestarved cells were sedimented and transferred to a complete minimal growth medium without proline. This stimulation of arginase activity was apparently due to the removal of proline, since transfer of the cells to a complete growth medium with added proline had no stimulatory effect. The stimulation of arginase was not prevented by addition of actinomycin D, and seemed therefore not to depend on the synthesis of new messenger RNA. An increased arginase activity was also found in cells starved for glutamine in presence of low concentrations of actinomycin D. In the experiment shown in Fig. 8 and Table IV cells were maintained for 18 hr in a glutamine free medium in presence of actinomycin D at concentrations ranging from 0.01 ,ug/ml to 0.2 pg/ml. The effects of these concentrations of actinomycin on the rate of RNA synthesis was determined by labeling samples from the cultures with 14C-uridine at the end of the starvation period. Since the differences in arginase accumulation during the experimental time could be due to unspecific effects on the overall rate of protein synthesis, this rate was deterExperimental
Cell Research
48
12
Eva Eliasson
mined by incorporation in Table
IV glutamine
starvation
which is in agreement ml of actinomycin
of 14C-leucine
2 hr after glutamine
in itself reduced
by 50 and 70 per cent respectively.
As seen
the rate of RNA synthesis,
with earlier results [S]. In presence
the rate of incorporation
addition.
of 0.01 and 0.025 pg/
of 14C-uridine was further reduced
In spite of the reduction
of the rate of RNA
;/ 0.6.
,.1’
,,1’ ,/’
HOURS
HOURS
Fig. 7.
Fig. 8.
Fig. 7.-Effect of actinomycin D. Cells were preincubated in glutamine-free medium for 22 hr. A. At 0 time was added: glutamine, 0.1 mM MnCl, and actinomycin at following concentrations pg/ml: 0 (o-o); 0.025 (V---V); 0.05 (n---a) and 0.1 (+---+). B. At 0 time glutamine was added (O-O), 5 hr later (indicated by arrow) 1 pg/ml actinomycin D (O---O). Experiment B conducted in the absence of manganese. A continuous destruction of arginase at a rate of 2-3 per cent/hr must therefore be taken in account. Fig. S.-Effect of actinomycin D. Cells were incubated for 18 hr in a glutamine-free medium containing 0.1 mM MnCl, and actinomycin D pg/ml: 0 (O-O); 0.01 ( +I-- +); 0.025 ( x --- x ); 0.05 ( n --- l ): 0.1 A --- A) and 0.2 ( V---v ). At 0 time glutamine was added. Incorporation of ‘%-u&dine was determined at the time of glutamine addition and incorporation of %Xeucine 2 hr later. cf. Table IV.
synthesis
the ability
measured actinomycin
of the cells to incorporate
2 hr after glutamine and only slightly
addition, reduced
14C-leucine
was not affected in the presence
into proteins, by 0.01
of 0.025
as
pug/ml of pg/ml. A
considerable stimulation of the specific arginase activity occurred in the presence of these concentrations of actinomycin D as seen in Fig. 8. (With higher concentrations of actinomycin, when the incorporation of 14C-uridine into RNA at the end of the starvation period decreased to < 6 per cent of the incorporation rate of normally growing cells, the rate of overall protein synthesis as well as the increase in arginase activity after glutamine addition was depressed. Since arginase accumulation was proportionally more affected than overall protein synthesis these results might be interpreted as an indication of formation of the messenger RNA for arginase synthesis during the Experimental
Cell Research 48
Repression
of arginase
synthesis
13
in Chang liver cells
glutamine starvation period. It seems however doubtful if such conclusions can be drawn from this experiment, because of the destruction of cells, which resulted in a net loss of 4-17 per cent of the protein content during the experimental time.) IV. Effect of actinomycin D on the rate of incorporation of 14C-uridine into RNA and on the rate of incorporation of “C-leucine into protein. (Same experiments as in Fig. 8.)
TABLE
The cells were incubated for actinomycin at concentrations
18 hr in a glutamine-free indicated.
medium, containing was then added.
Glutamine
Incorporation of labeled precursors in RNA and protein was proceeded The incorporation of r4C-uridine of the exponentially growing cells from ment was 2250 CPM/mg protein.
Culture (Fig. 8) A B C D E F
Actinomycin (,wdmU 0.01 0.025 0.05 0.1 0.2
D
Incorporation of %-uridine at the time glutamine addition (CPM/mg protein)
of
mM MnCl, and
0.1
as in Tables I and III. the start of the experi-
Incorporation of r4C-leucine 2 hr after glutamine addition (CPM/mg protein)
760 370 220 130 110 100
7900 8100 6800 5000 3900 2300
DISCUSSION
The present data indicate that the increase in arginase activity in prestarved Chang cells was due to a rapid synthesis of the enzyme. This increase was not prevented by the addition of actinomycin D at concentrations high enough for an almost complete inhibition of DNA directed RNA synthesis [5, 21, 321. The experimental data are consistent with a synthesis of arginase in response to a preformed messenger RNA. This messenger seemsto be stable at least for 4 hr. The rapid increase in arginase during the first hours after the restitution of the normal cellular environment was followed by a retardation of enzyme synthesis. This decrease in the rate of enzyme formation could be due to a destruction of the messenger RNA responsible for the initial increase in enzymic activity. However, experiments, showing that the decrease in rate of enzyme formation was prevented by the addition of actinomycin D, suggested that the retardation of enzyme synthesis in the absence of actinomycin might not be due to a destruction of messenger, but rather was the result of a decreased rate of translation of a stable messenger RNA. Experimental
Cell Research
48
Eva Eliasson
14
A regulation of arginase formation acting on the level of translation was also indicated by the results of experiments with cells prestarved for glutamine in the presence of proline. When such cells were spun down and transferred to a complete growth medium with added proline only a moderate increase in arginase occurred. If the cells were instead transferred to the same medium without proline a more rapid increase in enzymic activity took place. This increase in enzyme formation, apparently dependent on the removal of proline, was not prevented, but rather stimulated in presence of high concentrations of actinomycin D. Therefore the stimulation of enzyme formation seemed not to depend on new synthesis of arginase messenger, but rather to be connected with an increase in the rate of enzyme formation in response to preexisting messenger RNA. A stimulation of arginase accumulation was also seen in Chang cells, prestarved for glutamine in presence of concentrations of actinomycin D which only partially inhibited RNA synthesis and were without significant effect on the rate of overall protein synthesis during the experimental time. Increased enzymic activities in animal cells, in presence of actinomycin D have been repeatedly demonstrated [l, 7, 12, 17, 18, 23, 29, 301. Different explanations for the stimulatory effect of actinomycin D have been proposed. One possibility is that the degradation of short-lived messenger RNA’s might favour the translation of stable messengers by decreasing the competition for ribosomes or protein precursors. The present results showing a stimulation of arginase in presence of actinomycin D without any simultaneous alteration in the rate of overall protein synthesis, seem not to favour this hypothesis. Another possibility seems to be that the rate of translation of stable messenger RNA’s in animal cells is regulated by high molecular repressors, which are rapidly turned over, and which for their synthesis are dependent of DNA directed RNA synthesis [7, 12, 301. The present results are not incompatible with such a regulation mechanism. However, in view of other possible effects of actinomycin, apart from the inhibition of DNA directed RNA synthesis are not excluded. (Relevant in this [ll, 15, 22, 321 a It ernative explanations connection might be the rapid destruction of cytoplasmic RNA, which occurs in cultured cells in presence of actinomycin [32].) Arginase accumulation in Chang cells can be repressed in the presence of proline [6]. It was suggested that arginase synthesis is regulated by product repression in a manner analogous to the regulation of many bacterial enzymes [33] and that proline or alternatively A’-pyrroline 5-carboxylate, the immediate precursor of proline, is involved in the regulation of arginase synthesis as a co-repressor. A’-Pyrroline 5-carboxylate seems to be in a strategic posiExperimental
Cell Research
48
Repression
of arginase
synthesis
15
in Chang liver cells
tion for a regulator metabolite at a branch point of the reaction chain, and externally added proline could increase the intracellular concentration of since proline inhibits both A’-pyrroline 5A’-pyrroline 5-carboxylate, carboxylate reductase [ 191 and A’-pyrroline carboxylate dehydrogenase [ 281. The increase in arginase activity of cells prestarved for one essential amino acid in the absence of proline, occurred irrespective of which essential amino acid had been omitted during the starvation period. It is evident that the rapid increase in the rate of protein synthesis in the prestarved cells might lead to a decrease in the intracellular pool of low molecular protein precursors, especially if the ability of the cells to synthesize these precursors decreases during the starvation period. It is possible that ornithine 6-transaminase, the enzyme which catalyses the formation of A’-pyrroline 5-carboxylate from ornithine, might be rate limiting for the reaction sequence from arginine to proline (camp. [29]). The activity of ornithine d-transaminase has been shown to decrease during temporal amino acid starvation, and the restitution of the level of this enzyme upon addition of the missing nutrient is relatively slow1 [29]. It seems therefore reasonable to suggest that the increase in arginase in cells, prestarved in a medium lacking proline, might be due to a transient decrease in the intracellular amount of the low molecular metabolic repressor substance. The apparent discrepancy between the lack of an immediate effect of the addition of proline to cells at the time of nutritional restoration and the rapid stimulation of arginase synthesis when proline was removed at the time of nutritional “step up” suggests that A’-pyrroline 5-carboxylate rather than proline might be the metabolic repressor of arginase synthesis. Finally the present results showing a stimulation (insensitive to actinomycin D) of arginase synthesis in connection with the removal of proline suggest that the effect of the co-repressor is related to a regulation of arginase formation on the level of translation rather than on the level of messenger formation. The correlation of the variations of arginase activity of Chang cells to a regulation of enzyme synthesis on the level of translation does not exclude the existence of a superimposed regulation determining the amount of specific messenger present. The establishment, during liver differentiation, of an arginase level 50 times as high as the maximum level of the enzyme in Chang cells might involve other regulatory mechanisms as well. 1 The experiments on regulation of ornithine &transaminase (291 in cells prestarved for one essential amino acid differed from the present experiment with respect to the cells used for the start of the experiments. While the present experiments were always started with cells in rapid growth the former experiments were started with non-growing cells 4 days after the last dilution of the stock culture. In these experiments the ornithine Stransaminase activity, after one day of amino acid starvation, corresponded to less than 50 per cent of the enzyme activity in “exponentially growing cells”. Experimental
Cell
Research
48
Eva Eliasson
16
SUMMARY
A rapid increase in specific arginase activity occurred in Chang liver cells immediately after addition of the missing nutrient to cells preincubated in a medium from which one essential amino acid had been omitted. The rapid change in enzymic activity offered an opportunity for studying specific enzyme regulation during short time experiments. 2. The experimental results indicate that the increase in specific arginase activity, which could be immediately stopped by the addition of puromycin, was due to a synthesis of the enzyme more rapid than in normally growing cells. 2. Actinomycin D at concentrations sufficient to inhibit DNA directed RNA synthesis did not prevent the increase in arginase activity, suggesting that the synthesis of the enzyme took place in response to a preformed stable messenger RNA. 3. A few hours after the addition of the missing nutrient to a prestarved culture, arginase synthesis was again retarded. (a) This decrease in the rate of enzyme formation could be prevented by addition of actinomycin D. The decrease in the rate of enzyme synthesis in absence of actinomycin, therefore, might not be due to a destruction of the specific messenger RNA, but rather to a depression of the rate at which the stable arginase messenger was translated. (b) When proline was added to the culture medium the decrease in the rate of enzyme formation was enhanced. The experimental results suggest a regulation of arginase synthesis on the level of translation by means of an actinomycin sensitive repressor. The activity of this repressor seems to depend on the presence of a low molecular co-repressor, which accumulates in the cells in presence of proline. 4. When proline was added to the deficient medium during the starvation period, the increase in arginase activity immediately after the addition of the missing nutrient was partially prevented. (a) An increased rate of arginase formation was produced when these cells, prestarved in presence of proline, were transferred to a “normal” growth medium without added proline. (b) This stimulation of arginase synthesis, brought about by the removal of proline, was not prevented by the addition of actinomycin D and therefore seemed not to be dependent of a formation of new messenger RNA. These results again suggest a proline dependent repression of arginase synthesis at the level of translation of a preformed, stable messenger RNA. Experimental
Cell
Research
48
Repression
of arginase
synthesis
in Chang
liver
17
cells
Thanks are due to Associate Professor Tryggve Gustafson for valuable advice and encouragement. I am also grateful to Professor Harold Strecker for his critical reading of the manuscript. The expert technical assistanceof Miss K. Arvidsson, Miss U. Hammar and Miss E. Nor&r is gratefully acknowledged. This work was supported by grants from the Swedish Cancer Society and from the Swedish National Research Council. REFERENCES
1. 2. 3. 4. 5. 6. 7.
EAGLE, EAGLE,
G. R. and H., PIEZ,
ROBINSON, D. S., K. A., FLEISCHMAN,
Biochem. J. 93,100
(1964). V. I., J. Biol.
Chem. 234, 592 (1959). ELIASSON, E. E., Biochim. Biophys. Acta 97, 449 (1965). ~ Biochim. Biophys. Res. Comm. In press (1967). ELIASSON, E. E., BAUER, E. and HULTIN, T., J. Cell Biol. 33, 287 (1967). ELIASSON, E. E. and STRECKER, H. J., J. Biol. Chem. 241, 5757 (1966). GARREN, L. D., HOWELL, R. R., TOMKINS, G. RI. and CROCCO, R. M., Proc. N&Z. Acad. Sci. 52, R.
and
OYA~~A,
8. 9. 10. 11. 12. 13. 14. 15. 16.
1121
(1964). M. J. and Cox, B. P., J. Cell Biol. 29, 1 (1966). HANKS, J. H. and WALLACE, R. E., Proc. Sot. Esptt Biol. Med. 71, 196 (1949). HOLMBERG, B., Exptl Cell Res. 22, 406 (1961). HONIG, G. H. and RABINOVITZ, M., Science 149, 1504 (1965). KENNEY, F. T. and ALBRITTON, W. L., Proc. Natl Acad. Sci. 54, 1693 (1965). KLEIN, E., Exptt Cell Res. 21, 421 (1960). Ibid. 22, 226 (1961). LASZLO, J., MILLER, D. S., MCCARTY, K. S. and HOCHSTEIN, P., Science 151, LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. and RANDALL, R. J., J. Biol.
17. 18. 19. 20.
MCAUSLAN, B. R., Virology 21, 383 (1961). NITOWSKY, H., GELLER, S. and CASPER, R., Fed. Proc. 23, 556 (1964). PEISACH, J. and STRECKER, H. J., J. Biot. Chem. 237, 2255 (1962). PAUL, J., FOTTRELL, P. F., FRESHNEY, I., JONDORFF, W. R. and STRUTHERS,
GRIFFIN,
1007 (1966). Chem. 193,265
(1951).
Cancer 21. 22. 23. 24. 25. 26. 27.
Inst.
Monograph
REICH,
R., FRANKLIN, R. M., SHATKIN, A. J. and TATUM, (1962). REVEL, M. and HIATT, H. H., Science 146,131l (1964). ROSEN, F., RAINA, P. N., MILHOLLAND, R. J. and NI~HOL, SCHIMKE, R. T., Biochim. Biophys. Acta 62, 599 (1962). ~ .J. Biol. Chem. 239, 136 (1964). . ’
~
Nail
SCHIMKE,
Cancer R.
Inst.
T., SWEENEY,
M.
G.,
Nat1
13, 219 (1964).
Monograph E. W.
E. L., Proc.
C. A.,
13, 197 (1964). BERLIN, C. M., Biochem.
and
Nat1 Acad.
Science
Biophys.
Sci. 48,123s
146, 661 (1964).
Res. Comm.
15, 214
(1964). 28. 29. 30. 31. 32. 33.
STRECKER, H. J., Biol. Chem. 235, 3218 (1960). STRECKER, H. J. and ELIASSON, E. E., J. Biol. Chem. 241, 5750 (1966). TOMSON, E. B., TOMKINS, G. M. and CURRAN, J. F., Proc. Nat1 Acad. Sci. 56, 296 (1966). TRAKATELLIS, A. C., AXELROD, A. E. and MONTJAR, M., Nature 203,1134 (1964). WIESNER, R., Acs, G., REICH, E. and SHAFIQ, A., J. Cell Biol. 27, 47 (1965). VOGEL, H. J. in D. M BONNER (ed.), Control Mechanisms in Cellular Processes, p. 23. Ronald, New York, 1961.
2-671819
Experimental
Cell Research
48
by Academic
Experimental
Press
Inc.
Cell Research
48, 1-17
REPRESSION
1
(1967)
OF ARGINASE SYNTHESIS CHANG LIVER CELLS EVA
The
Wenner-Gren
Institute,
IN
ELIASSON
University
of Stockholm,
Stockholm,
Sweden
Received June 1, 1967
VARIATIONSin
arginase activity in cultured animal cells related to variations in the composition of the growth medium have been demo&rated repeatedly [3, 6, 13, 14, 20, 24, 25, 261. In an established cell strain grown in this laboratory (Chang liver) arginase activity varies at least 40-fold, from 0.05 units/mg protein [3] to 2.0 units/mg protein [6], under different growth conditions. These variations are readily reversible, and seem not to be due to a selection of cells with a different genetic constitution. Results from experiments with Chang cells also suggest that arginase activity is repressed by a product of a metabolic pathway initiated by the arginase reaction [3, 61. However, definite conclusions as to the mechanism of this regulation, which in its effect is analogous to product repression in bacteria, could not be drawn from these experiments, The aim of the present investigation was to get further information about the mechanism of regulation of arginase activity in cultured cells. Preliminary experiments with Chang cells had shown that temporary omission of a single essential amino acid from the minimal growth medium resulted in a rapid increase in the specific arginase activity of these cells, immediately after addition of the missing amino acid. This increase in enzymic activity, which was large enough to be measured within 1 hr, offered an opportunity for studying the regulation of enzyme accumulation during short time experiments. The advantages of such experiments were several: (1) The continuous changes in the environment of the cells, occurring in stationary cultures as a result of the metabolism of the cells, were less extensive during short periods of time. (2) The degradation of the enzyme was small as compared with its synthesis during the experimental time. Differences in the rate of accumulation could therefore be ascribed to differences in the rate of synthesis, rather than to differences in rate of destruction of the enzyme. (3) The effect on specific enzyme synthesis of an inhibition of DNA dependent RNA synthesis by addition of actinomycin D, required study over short periods of time, because of the general toxic effects of high concentra1 - 671819
Experimental
Cell Research
48
2
Eva Eliasson
tions of actinomycin D on cultured cells. These effects of the antibiotic were especially pronounced when the cells were grown in suspension cultures [S, 321, and resulted in an irreversible loss of viability and a rapid decrease in the overall content of protein and RNA of the cells. MATERIALS
AND
METHODS
Chang liuer cells originally obtained from Microbiological Associates, Bethesda, Maryland, were cultivated in suspension in a slightly modified Eagle medium supplemented with 10 per cent horse serum as previously reported [29]. The stock cultures, from which single experiments were started, were propagated through serial dilution with fresh medium twice a week to a cell concentration of 0.25 x lo6 cells/ml. The present experiments were started with cells from such stock cultures 48-66 hr after the last dilution. The cells, which were then in a state of rapid growth with a generation time. of 30-35 hr, were sedimented and transferred to fresh medium at a cell density of 0.5-0.7 x 106 cells/ml. In spite of the care taken to standardize the culture methods, it was impossible to avoid variations in the arginase activity of the stock cultures [6]. Comparable experiments were therefore always started with cells from the same stock culture and with serum from the same batch. Testing for contaminations were conducted as previously described [3]. No contaminations were found. The viability of the cells was determined by means of the Lissamine green permeability test [lo]. Cell counts were made either microscopically with aid of a Btirker counting chamber or in a Ljungberg celloscope 101 (AB Lars Ljungberg and Co., Stockholm). Total cell protein was determined according to Lowry el al. [16]. The cells were collected by centrifugation at 500 g for 7 min, washed with cold Hanks’ salt solution [9] and resuspended in distilled water. The determinations of total protein content were in agreement with results from the cell counts, indicating that the preparation for protein determination did not remove protein from the cells. Determination of arginase activity has been described [6] and specific enzyme activity is expressed as pmoles of urea formed per hour and mg protein. Determination of the rate of incorporation of labeled precursors into protein and RNA has also been described [5]. Samples from the cultures were incubated in media containing 0.4 mM W-L-leucine (2 mC/mmole) for IO min or 0.2 mM 14C-uridine (4 mC/mmole) for 20 min. ChemicaZs.--‘4C-labeled leucine was purchased from Radiochemical Centre, Amersham, England, 14C-2-uridine from New England Nuclear Corp., Boston, Mass., actinomycin D from Merck, Sharp and Dohme Laboratories and puromycin dihydrochloride was obtained from the Nutritional Biochemicals Corp., Cleveland 28, Ohio. RESULTS
The effect of temporary
amino
acid deficiency
on arginase
activity
Exponentially growing Chang cells were sedimented and transferred to a minimal medium from which one essential amino acid had been omitted. 0.1 mM MnCl, was included in the medium in order to stabilize arginase Experimental
Cell Research
48
Repression
of arginase
synthesis
in Chang liver cells
3
[S, 261. On the next day the missing nutrient was added to the culture at a concentration corresponding to the normal growth medium. The viability of the cells was not impaired by maintenance in a deficient medium for one day, and the normal rate of protein synthesis was restored within 2-4 hr after addition of the missing nutrient (cf. [5]). The increase in specific arginase activity after addition of glutamine or phenylalanine to cultures maintained for one day in deficient media is shown in Fig. 1. A similar increase in enzymic activity after starvation for arginine or histidine is seen in Fig. 4A (open symbols). Essentially the same effect was obtained if the prestarved cells were sedimented and transferred to a “normal” growth medium. This rapid increase in arginase activity in prestarved cells on addition of the missing nutrient occurred irrespective of which amino acid had been omitted during the starvation period, indicating that the increase of enzymic activity might be an unspecific effect of nutritional “step up” conditions after amino acid deprival. The rate of increase in arginase activity depended on the length of the starvation period as shown in Fig. 2. This experiment was conducted with parallel cultures in presence or absence of manganese and the cells were prestarved for glutamine for 0, 15 and 24 hr. The increase the enzyme
in arginase
activity-a
result of an increased
rate of synthesis
of
The immediate increase in arginase in prestarved cells restored nutritionally was prevented by the concomitant addition of puromycin (10 ,ug/ml), in-
I
10
.! 20 30 HOWlS
Fig.
1.
40
so
I
10
20
30
HOURS
Fig.
2.
Fig. l.-The effect on specific arginase activity of temporal starvation for glutamine or phenylalanine. Chang cells were incubated for 23 hr in media lacking glutamine (O-O) or phenylalanine (A-A). At 23 hr the missing amino acids were added. The growth media contained 0.1 mM MnCl,. Fig. 2.-The increase of arginase activity after glutamine addition to cells preincubated in a glutamine-free medium for 0, 15 and 24 hr. Cells were incubated in a glutamine-free medium in presence ( 00) and in absence (AA) of 0.1 mM MnCl,. At times indicated by arrows glutamine was added. Specific arginase activity 3 hr after glutamine addition denoted by filled symbols. Experimental
Cell
Research
48
Eva Eliasson dicating that synthesis of enzyme protein rather than an activation of preformed enzyme took place. If the enzyme were rapidly turning over in normally growing cells, an accumulation of newly formed enzyme molecules might be, however, the result merely of a stabilization of the enzyme protein [27]. It has been shown that arginase in HeLa cells turns over more rapidly than the average cell protein [26]. Although this seems to be true also for arginase in Chang cells the following experiment indicated that the increase in arginase at nutritional “step up” was due to an acceleration of the rate, at which the enzyme was synthesized rather than to a stabilization effect. In the experiment shown in Fig. 3 and Tables I and II duplicate cultures were started with cells from three stock cultures with different specific arginase activities. (The low activity of the stock culture from which cultures E and F in Fig. 3 were started was due to the addition of 6 mM proline to the growth medium for 10 days before the start of the experiment (cf. [6]). The cells were incubated in a glutamine-free medium for 20 hr. Glutamine was then added, and at the same time 0.1 mM MnCl,, to cultures B, D and F (open symbols).) Protein determinations indicated that the growth of the cells during the 24 hr period after the addition of glutamine was not significantly affected by the presence of manganese (Fig. 3 B). The labeling data in Table I show that the rate of ‘“C-leucine incorporation into protein at the end of the starvation period was 10 per cent of the rate of incorporation into exponentially growing cells. The rate of overall protein synthesis was, however, rapidly restored on addition of glutamine, and there was no significant difference in the rate of incorporation of 14C-leucine between the cultures to which manganese had been added and the corresponding control cultures. Differences in the increase in specific arginase activity between the cultures were therefore not due to differences in overall growth. During the first four hours after glutamine addition the rapid increase in arginase activity (Fig. 3A) occurred at about the same rate in all cultures, though manganese always had a slightly stimulatory effect. During the period 24 to 43 hr, i.e. 4 to 23 hr after glutamine addition, specific arginase activity decreased (A) or increased only slightly (E) in the absence of manganese. In presence of manganese there was a considerable increase in arginase activity during this period also, but this increase was always less rapid than during the first four hours after glutamine addition. This was true not only for the increase in specific activity but also for the increase in total activity of the I whole culture. A comparison between the development of the enzyme activity Experimental
Cell Research
48
in cultures
Repression
of arginase
synthesis
in Chang liver cells
5
B and F (grown in presence of manganese) indicates that the increase of enzymic activity in these cultures was independent of the amount of enzyme present in the cells. It seems reasonable, therefore, to conclude that destruction of the enzyme was not a determining factor for the development of the enzyme in the presence of manganese. The stability of arginase in the presence of manganese is in good agreement with earlier results from experiments with non-growing Chang cells [6] and with HeLa cells [26]. TABLE
I. Incorporation
rate of 14C-L-leucine into protein of cells from the experiment shown in Fig. 3.
For incorporation 2.5 x lo6 cells were sedimented and resuspended in 275 ~1 medium containing 0.4 m&f %-L-leucine (2 mC/mmole) and incubated for 10 min at 37°C.
Culture (Fig. 3) C
Time (hours from start) 0
Treatment Exponentially culture
20
20 hr glutamine
22
20 2 20 2
22
24 24
20 4 20 4
hr glutamine hr complete hr glutamine hr complete + MnCl, hr glutamine hr complete hr glutamine hr complete + MnCl,
of the cells
Incorp. of 14Cleucine in 10 min (CPM/w? protein)
growing stock
8000
starvation
900=
starv. medium starv. medium
7200
starv. medium starv. medium
7400
starv.
1000=
8000
7600
E
20
20 hr glutamine
E
22 22
hr glutamine starv. hr complete medium hr glutamine starv. hr complete medium + MnCl,
8500
F
20 2 20 2
E
24 24
hr glutamine starv. hr complete medium hr glutamine starv. hr complete medium + MnCl,
9400
F
20 4 20 4
cells were labeled in a glutamine-free
medium.
a Glutamine-deficient
9400
8500
Experimental
Cell Research
48
6
Eva Eliasson
In the absence of manganese the accumulation of arginase was always less rapid than in its presence. It seemed reasonable to believe that the difference in enzyme accumulation between cultures A and B, C and D or E and F respectively was due to the destruction of the enzyme in absence of manganese (camp. [26]). On th’ is assumption the destruction of enzyme, per unit enzyme present, per hour was calculated (Table II). The results indicate a destruction rate of arginase of 2 to 3 per cent/hr during the period 23 [24] to 43 hr, which is higher than the turnover rate for average cell protein in cultured cells of II. Effect of manganese on arginase accumulation in cultures started with cells with different specific activities (Fig. 3).
TABLE
Fraction Time W
Specific act.
Mean value Culture
20
of enzyme
Increase A
23
0.66
+ 0.20
-0.11
0.55 C
+ 0.25
E
0.34
= 0.02a
= 0.02
0.61 x 20 (Increase D) - (Increase (Sp. act. C) x (time) 0.31 - 0.25
F (MrPf)
0.46 x 4
C)
= 0.03a
(Increase F) - (Increase (Sp. act. E) x (time) 0.23 - 0.21
0.13 + 0.21
+ 0.23
0.24 x 4
E)
= 0.02a
0.36 0.26 - 0.07
0.36 0.38
43
D (Mn2+)
+ 0.31
Culture
0.24 0.34
0.56 x 3
0.64
0.13
24
0.24 - 0.20
0.17 t 0.11
0.33
0.58
24
+ 0.24
+ 0.17
Culture
0.33
Culture 20
A)
0.87
0.46 24
(Increase B) - (Increase (Sp. act. A) x (time)
B (Mns+)
0.70
Culture 20
Increase
of MnCl,.
0.70
0.61 43
Specific act.
0.46 0.56
0.66
in 1 hr in the absence
Culture
0.46
23
inactivated
+ 0.07
0.41
+ 0.26
0.38 x 19
= 0.03
0.62
a The difference in the increase in arginase in presence or in absence of Mn*+ during the short interval of 3 or 4 hr is near the limit of the accuracy of the determinations, but -admits a rough estimation of the rate of destruction of the enzyme. The results are in good agreement with the results of several other similar experiments. Experimental
Cell Research
48
Repression
of arginase
synthesis
7
in Chang liver cells
1 per cent/hr as determined by Eagle et al. [2]. The calculations of the destruction rate of the enzyme during the short period of rapid increase in enzyme activity during the first 3 or 4 hr after glutamine addition might be less accurate, because of the small differences between the activities attained within the short time interval, but the results indicate that the destruction of .*
HOURS
B
I 10
: 20
30 HOURS
Fig. 3.
I LO
. SO
I 10
20
30
40
50
HOURS
Fig. 4.
Fig. 3.-A, The effect of 0.1 mM MnCl, on arginase activity of cells prestarved for glutamine. Cultures A ( 0 - - - 0 ) and B ( O-o) were started with exponentially growing cells from the same stock culture. Cultures C (A - - -A) and D ( n--n) from another stock culture. Cultures E ( n - -- n ) and F ( q - q ) were started from a serially diluted culture, which had grown in presence of 6 mM proline for 10 days, and had therefore a low arginase activity. The cells were incubated in a glutamine-free medium without additions for 20 hr. Glutamine was then added and at the same time 0.1 mM MnCl, to cultures B, D and F (open symbols). B, Growth of cultures (symbols as in A); pg cell protein per ml culture. Fig. 4.-A, The effect of 6 m&f proline present in the culture medium during temporary amino acid starvation and subsequent nutritional restoration. The cells were incubated in media without arginine ( 0 - 0, w - -- w ) or histidine (AA, A---A). 23 hr thereafter the missing amino acid was added. The media contained 0.1 mM MnCl, and the media of the cultures indicated by filled symbols also 6 mM proline. B, The lack of immediate effect of 6 mM proline added together with the missing amino acid. Cells were incubated for 23 hr in a glutamine-free medium, containing 0.1 m&f MnCl,. At 23 hr glutamine (O-O) or glutamine + 6 m&f proline ( 0 -- - 0) was added.
the enzyme in the absence of manganese occurred at about the same rate during the whole experimental time. Under no conditions did the turnover rate of the enzyme appear to be high enough to account for the rapid increase in enzyme activity during the first hours after glutamine addition solely as a result of stabilization. It seems therefore reasonable to believe that the accumulation of arginase under Experimental
Cell Research
48
Eva Eliasson
8 nutritional
“step up” conditions
was due to a high rate of synthesis
of the
enzyme protein. Repression of arginase synthesis in the presence of proline The reaction
sequence to proline by way of A’-pyrroline
been suggested to be a major route for the metabolism
HOURS
Fig. 5.
5-carboxylate,
of ornithine,
has
derived
hOuRs
Fig. 6.
Fig. 5.-A. The effect on arginase activity of proline present during glutamine starvation and subsequent nutritional restoration. The cells were incubated for 21 hr in a glutamine-free medium with 6 mM proline ( 0 - - - 0 ) or without proline addition ( O- 0). At 21 hr glutamine was added. B. The effect of proline added together with glutamine to a glutamine deficient culture. Cells were incubated for 19 hr in a glutamine-free medium. At 19 hr glutamine (O-O) or glutamine + 6 mM proline was added ( 0 --- 0 ). The experiments were conducted in the absence of manganese. A continuous destruction of arginase at a rate of 2-3 per cent/hr must be taken in account when comparing the results with the results of the’ experiments of Fig. 4. Fig. B.-Effect of removal of proline at the time of glutamine addition to a culture prestarved for glutamine in presence of 6 mM proline. The cells were maintained in a glutamine-free medium in presence of 6 m&f proline for 24 hr. The cells were then spun down and transferred to a complete, minimal growth medium without proline (O-O) or to the same medium with 1 pg/ml of actinomycin D (a... n) or to a complete medium with 6 m&f proline added (O---O) (no manganese present, see legend Fig. 5).
from arginine via arginase, in mammalian cell strains [6, 291. Recent results have shown that arginase activity was repressed in Chang liver cells when proline was added to the minimal growth medium [6]. A series of experiments were conducted in order to investigate the effect of proline added to cell cultures at different times during amino acid starvation and subsequent nutritional “step up”. During the starvation period (s.f. [4]) as well as during the period later than 4 to 6 hr after nutritional “step up”, proline had always a repressing the arginase activity (Figs. 4 A and 5). Experimental
Cell Research 48
effect on
Repression
of arginase
synthesis
9
in Chang liver cells
During the first hours after the addition of the missing amino acid to prestarved cells, proline repressed arginase activity only if present already during the starvation period (Figs. 4 and 5). (If glutamine was the amino acid omitted during the starvation period the increase in arginase at “step up” was only partially repressed, while the effect of proline was more pronounced in cells prestarved for arginine or histidine.) When cells prestarved for glutamine in the presence of proline were spun down and transferred to a complete growth medium lacking proline the repression of arginase synthesis was immediately reversed (Fig. 6). Addition of 6 mM proline to the growth medium seemed to affect arginase accumulation specifically, since it had no significant effect on viability and overall growth of the cells. Nor did added proline affect the rate of incorporation of 14C-labeled leucine into cellular protein or the incorporation of 14Curidine into RNA. On the whole the results are compatible with earlier results [6] indicating that the rate of arginase synthesis in Chang cells is specifically correlated to the intracellular level of a metabolic repressor. Experiments with actinomycin gave some further information about the localization of the mechanism responsible for the regulation of arginase synthesis. A stable messenger
for arginase
synthesis
Recent results [5] have shown that a restoration of overall protein synthesis in glutamine starved Chang cells takes place within a few hours after addition of the missing amino acid. This restitution of protein synthesis occurs without a preceding increase in the prevailing low rate of RNA synthesis and is not prevented by addition of actinomycin D. It was therefore suggested that previously synthesized messenger RNA is brought into action in response to the reestablishment of an adequate cellular environment. In the two experiments shown in Table III actinomycin D (0.5 or 1 pg/ml) was added to cell cultures which had been maintained in a glutamine-free medium for 23 hr. One hour later glutamine was added. In view of the rapid inhibitory effect of actinomycin D on RNA synthesis of cultured cells [5, 21, 321 it seems reasonable to believe that RNA synthesis was effectively inhibited in the experimental cells already at the time of glutamine addition. (Four hours after the addition of glutamine the incorporation of 14C-uridine into RNA had decreased to 1 per cent of the rate in “normally” growing Chang cells.) In spite of the inhibition of RNA synthesis arginase activity increased by SO-100 per cent during a 4 hr period after the addition of glutamine. This Experimental
Cell
Research
48
10
Eva Eliasson
increase occurred at about the same rate as in the control cells in the absence of actinomycin D. These results strongly suggest that the arginase formation in “step up” cells took place in response to previously formed messenger RNA, and that this messenger had a life time exceeding 4 hr. Identical results were also attained with cells prestarved of histidine. III.
TABLE
Effect of actinomycin increase
The cells were incubated then added. Actinomycin For incorporation medium containing
Experiment I
II
D on the rate of RNA-synthesis in arginase activity.
in a glutamine-free medium (no manganese) for 24 hr. D was added 1 hr before the addition of glutamine.
of W-uridine 2.5 x lo6 cells were 0.2 mM r4C-uridine (4 mC/mmole)
Treatment
of the cells
Exponentially growing cells 24 hr glutamine starvation 24 hr glutamine starv., 4 hr complete medium 24 hr glutamine starv., 4 hr complete medium + actinomycin 24 hr glutamine starv. 24 hr glutamine starv., 4 hr complete medium 24 hr glutamine starv., 4 hr complete medi1.m i- actinomycin
and on the Glutamine
was
sedimented and resuspended in 275 and incubated for 20 min at 37°C. Incorp. of W-uridine (CPM/w? protein)
Sp. arginase act.
-
2510 740 590
0.31 0.23 0.46
1
20
0.46
-
630 690
0.37 0.70
30
0.67
Actinomycin ha/ml)
D
,ul
D
0.5 D
(If similar experiments were extended over longer periods of time a decrease in the rate of arginase accumulation occurred in the presence of these concentrations of actinomycin D. However, conclusions as to the life time of the arginase messenger could not be drawn from such experiments because of a general cell destruction, which resulted in gross morphological changes, loss of viability and a decrease in the total cellular RNA and protein.) When lower concentrations of actinomycin D (0.025-0.1 ,ug/ml) were used in similar experiments arginase accumulation during the first 5 hr after nutritional “step up” was not affected. After that time, when the increase in arginase activity in the control cultures was normally retarded, arginase activity was not decreased but on the contrary slightly stimulated in the presence of actinomycin D (Fig. 7A). Experimental
Cell Research
48
Repression Regulation messenger
of arginase
of the rate of arginase RNA
synthesis synthesis
11
in Chang liver cells on the level of translation
of a stable
In order to investigate further the effect on arginase formation of an inhibition of RNA synthesis at the time when enzyme synthesis was normally were performed. retarded in the “step up” cells a series of experiments Actinomycin D at concentrations of 0.5 or 1 ,ug/ml was added 5 hr after the addition of glutamine to prestarved cultures. The result of one of these experiments, which all gave similar results, is shown in Fig. 7 B. (The experiment was made in the absence of manganese and a continuous destruction of enzyme during the experimental time must be taken in account.) The stimulation of enzymic activity by addition of actinomycin D at concentrations high enough to inhibit most of the DNA dependent RNA synthesis, strongly suggested that the decrease in the rate of enzyme formation in the absence of actinomycin D was not due to a decrease in the amount of messenger RNA present, but rather to a decrease in the rate at which a stable messenger was translated in to enzyme protein. An alternative explanation, which cannot be excluded on the basis of these experiments, could be an inhibition of messenger break down in presence of actinomycin D (camp. [l, 311). However, further support for the hypothesis postulating a regulation of the rate of arginase formation at the level of translation of a stable messenger RNA was attained from the experiments, shown in Fig. 6. When glutamine was added to a cell culture prestarved of glutamine for 24 hr in presence of 6 mM proline the increase in arginase was comparatively slow. A stimulation of arginase activity occurred if the prestarved cells were sedimented and transferred to a complete minimal growth medium without proline. This stimulation of arginase activity was apparently due to the removal of proline, since transfer of the cells to a complete growth medium with added proline had no stimulatory effect. The stimulation of arginase was not prevented by addition of actinomycin D, and seemed therefore not to depend on the synthesis of new messenger RNA. An increased arginase activity was also found in cells starved for glutamine in presence of low concentrations of actinomycin D. In the experiment shown in Fig. 8 and Table IV cells were maintained for 18 hr in a glutamine free medium in presence of actinomycin D at concentrations ranging from 0.01 ,ug/ml to 0.2 pg/ml. The effects of these concentrations of actinomycin on the rate of RNA synthesis was determined by labeling samples from the cultures with 14C-uridine at the end of the starvation period. Since the differences in arginase accumulation during the experimental time could be due to unspecific effects on the overall rate of protein synthesis, this rate was deterExperimental
Cell Research
48
12
Eva Eliasson
mined by incorporation in Table
IV glutamine
starvation
which is in agreement ml of actinomycin
of 14C-leucine
2 hr after glutamine
in itself reduced
by 50 and 70 per cent respectively.
As seen
the rate of RNA synthesis,
with earlier results [S]. In presence
the rate of incorporation
addition.
of 0.01 and 0.025 pg/
of 14C-uridine was further reduced
In spite of the reduction
of the rate of RNA
;/ 0.6.
,.1’
,,1’ ,/’
HOURS
HOURS
Fig. 7.
Fig. 8.
Fig. 7.-Effect of actinomycin D. Cells were preincubated in glutamine-free medium for 22 hr. A. At 0 time was added: glutamine, 0.1 mM MnCl, and actinomycin at following concentrations pg/ml: 0 (o-o); 0.025 (V---V); 0.05 (n---a) and 0.1 (+---+). B. At 0 time glutamine was added (O-O), 5 hr later (indicated by arrow) 1 pg/ml actinomycin D (O---O). Experiment B conducted in the absence of manganese. A continuous destruction of arginase at a rate of 2-3 per cent/hr must therefore be taken in account. Fig. S.-Effect of actinomycin D. Cells were incubated for 18 hr in a glutamine-free medium containing 0.1 mM MnCl, and actinomycin D pg/ml: 0 (O-O); 0.01 ( +I-- +); 0.025 ( x --- x ); 0.05 ( n --- l ): 0.1 A --- A) and 0.2 ( V---v ). At 0 time glutamine was added. Incorporation of ‘%-u&dine was determined at the time of glutamine addition and incorporation of %Xeucine 2 hr later. cf. Table IV.
synthesis
the ability
measured actinomycin
of the cells to incorporate
2 hr after glutamine and only slightly
addition, reduced
14C-leucine
was not affected in the presence
into proteins, by 0.01
of 0.025
as
pug/ml of pg/ml. A
considerable stimulation of the specific arginase activity occurred in the presence of these concentrations of actinomycin D as seen in Fig. 8. (With higher concentrations of actinomycin, when the incorporation of 14C-uridine into RNA at the end of the starvation period decreased to < 6 per cent of the incorporation rate of normally growing cells, the rate of overall protein synthesis as well as the increase in arginase activity after glutamine addition was depressed. Since arginase accumulation was proportionally more affected than overall protein synthesis these results might be interpreted as an indication of formation of the messenger RNA for arginase synthesis during the Experimental
Cell Research 48
Repression
of arginase
synthesis
13
in Chang liver cells
glutamine starvation period. It seems however doubtful if such conclusions can be drawn from this experiment, because of the destruction of cells, which resulted in a net loss of 4-17 per cent of the protein content during the experimental time.) IV. Effect of actinomycin D on the rate of incorporation of 14C-uridine into RNA and on the rate of incorporation of “C-leucine into protein. (Same experiments as in Fig. 8.)
TABLE
The cells were incubated for actinomycin at concentrations
18 hr in a glutamine-free indicated.
medium, containing was then added.
Glutamine
Incorporation of labeled precursors in RNA and protein was proceeded The incorporation of r4C-uridine of the exponentially growing cells from ment was 2250 CPM/mg protein.
Culture (Fig. 8) A B C D E F
Actinomycin (,wdmU 0.01 0.025 0.05 0.1 0.2
D
Incorporation of %-uridine at the time glutamine addition (CPM/mg protein)
of
mM MnCl, and
0.1
as in Tables I and III. the start of the experi-
Incorporation of r4C-leucine 2 hr after glutamine addition (CPM/mg protein)
760 370 220 130 110 100
7900 8100 6800 5000 3900 2300
DISCUSSION
The present data indicate that the increase in arginase activity in prestarved Chang cells was due to a rapid synthesis of the enzyme. This increase was not prevented by the addition of actinomycin D at concentrations high enough for an almost complete inhibition of DNA directed RNA synthesis [5, 21, 321. The experimental data are consistent with a synthesis of arginase in response to a preformed messenger RNA. This messenger seemsto be stable at least for 4 hr. The rapid increase in arginase during the first hours after the restitution of the normal cellular environment was followed by a retardation of enzyme synthesis. This decrease in the rate of enzyme formation could be due to a destruction of the messenger RNA responsible for the initial increase in enzymic activity. However, experiments, showing that the decrease in rate of enzyme formation was prevented by the addition of actinomycin D, suggested that the retardation of enzyme synthesis in the absence of actinomycin might not be due to a destruction of messenger, but rather was the result of a decreased rate of translation of a stable messenger RNA. Experimental
Cell Research
48
Eva Eliasson
14
A regulation of arginase formation acting on the level of translation was also indicated by the results of experiments with cells prestarved for glutamine in the presence of proline. When such cells were spun down and transferred to a complete growth medium with added proline only a moderate increase in arginase occurred. If the cells were instead transferred to the same medium without proline a more rapid increase in enzymic activity took place. This increase in enzyme formation, apparently dependent on the removal of proline, was not prevented, but rather stimulated in presence of high concentrations of actinomycin D. Therefore the stimulation of enzyme formation seemed not to depend on new synthesis of arginase messenger, but rather to be connected with an increase in the rate of enzyme formation in response to preexisting messenger RNA. A stimulation of arginase accumulation was also seen in Chang cells, prestarved for glutamine in presence of concentrations of actinomycin D which only partially inhibited RNA synthesis and were without significant effect on the rate of overall protein synthesis during the experimental time. Increased enzymic activities in animal cells, in presence of actinomycin D have been repeatedly demonstrated [l, 7, 12, 17, 18, 23, 29, 301. Different explanations for the stimulatory effect of actinomycin D have been proposed. One possibility is that the degradation of short-lived messenger RNA’s might favour the translation of stable messengers by decreasing the competition for ribosomes or protein precursors. The present results showing a stimulation of arginase in presence of actinomycin D without any simultaneous alteration in the rate of overall protein synthesis, seem not to favour this hypothesis. Another possibility seems to be that the rate of translation of stable messenger RNA’s in animal cells is regulated by high molecular repressors, which are rapidly turned over, and which for their synthesis are dependent of DNA directed RNA synthesis [7, 12, 301. The present results are not incompatible with such a regulation mechanism. However, in view of other possible effects of actinomycin, apart from the inhibition of DNA directed RNA synthesis are not excluded. (Relevant in this [ll, 15, 22, 321 a It ernative explanations connection might be the rapid destruction of cytoplasmic RNA, which occurs in cultured cells in presence of actinomycin [32].) Arginase accumulation in Chang cells can be repressed in the presence of proline [6]. It was suggested that arginase synthesis is regulated by product repression in a manner analogous to the regulation of many bacterial enzymes [33] and that proline or alternatively A’-pyrroline 5-carboxylate, the immediate precursor of proline, is involved in the regulation of arginase synthesis as a co-repressor. A’-Pyrroline 5-carboxylate seems to be in a strategic posiExperimental
Cell Research
48
Repression
of arginase
synthesis
15
in Chang liver cells
tion for a regulator metabolite at a branch point of the reaction chain, and externally added proline could increase the intracellular concentration of since proline inhibits both A’-pyrroline 5A’-pyrroline 5-carboxylate, carboxylate reductase [ 191 and A’-pyrroline carboxylate dehydrogenase [ 281. The increase in arginase activity of cells prestarved for one essential amino acid in the absence of proline, occurred irrespective of which essential amino acid had been omitted during the starvation period. It is evident that the rapid increase in the rate of protein synthesis in the prestarved cells might lead to a decrease in the intracellular pool of low molecular protein precursors, especially if the ability of the cells to synthesize these precursors decreases during the starvation period. It is possible that ornithine 6-transaminase, the enzyme which catalyses the formation of A’-pyrroline 5-carboxylate from ornithine, might be rate limiting for the reaction sequence from arginine to proline (camp. [29]). The activity of ornithine d-transaminase has been shown to decrease during temporal amino acid starvation, and the restitution of the level of this enzyme upon addition of the missing nutrient is relatively slow1 [29]. It seems therefore reasonable to suggest that the increase in arginase in cells, prestarved in a medium lacking proline, might be due to a transient decrease in the intracellular amount of the low molecular metabolic repressor substance. The apparent discrepancy between the lack of an immediate effect of the addition of proline to cells at the time of nutritional restoration and the rapid stimulation of arginase synthesis when proline was removed at the time of nutritional “step up” suggests that A’-pyrroline 5-carboxylate rather than proline might be the metabolic repressor of arginase synthesis. Finally the present results showing a stimulation (insensitive to actinomycin D) of arginase synthesis in connection with the removal of proline suggest that the effect of the co-repressor is related to a regulation of arginase formation on the level of translation rather than on the level of messenger formation. The correlation of the variations of arginase activity of Chang cells to a regulation of enzyme synthesis on the level of translation does not exclude the existence of a superimposed regulation determining the amount of specific messenger present. The establishment, during liver differentiation, of an arginase level 50 times as high as the maximum level of the enzyme in Chang cells might involve other regulatory mechanisms as well. 1 The experiments on regulation of ornithine &transaminase (291 in cells prestarved for one essential amino acid differed from the present experiment with respect to the cells used for the start of the experiments. While the present experiments were always started with cells in rapid growth the former experiments were started with non-growing cells 4 days after the last dilution of the stock culture. In these experiments the ornithine Stransaminase activity, after one day of amino acid starvation, corresponded to less than 50 per cent of the enzyme activity in “exponentially growing cells”. Experimental
Cell
Research
48
Eva Eliasson
16
SUMMARY
A rapid increase in specific arginase activity occurred in Chang liver cells immediately after addition of the missing nutrient to cells preincubated in a medium from which one essential amino acid had been omitted. The rapid change in enzymic activity offered an opportunity for studying specific enzyme regulation during short time experiments. 2. The experimental results indicate that the increase in specific arginase activity, which could be immediately stopped by the addition of puromycin, was due to a synthesis of the enzyme more rapid than in normally growing cells. 2. Actinomycin D at concentrations sufficient to inhibit DNA directed RNA synthesis did not prevent the increase in arginase activity, suggesting that the synthesis of the enzyme took place in response to a preformed stable messenger RNA. 3. A few hours after the addition of the missing nutrient to a prestarved culture, arginase synthesis was again retarded. (a) This decrease in the rate of enzyme formation could be prevented by addition of actinomycin D. The decrease in the rate of enzyme synthesis in absence of actinomycin, therefore, might not be due to a destruction of the specific messenger RNA, but rather to a depression of the rate at which the stable arginase messenger was translated. (b) When proline was added to the culture medium the decrease in the rate of enzyme formation was enhanced. The experimental results suggest a regulation of arginase synthesis on the level of translation by means of an actinomycin sensitive repressor. The activity of this repressor seems to depend on the presence of a low molecular co-repressor, which accumulates in the cells in presence of proline. 4. When proline was added to the deficient medium during the starvation period, the increase in arginase activity immediately after the addition of the missing nutrient was partially prevented. (a) An increased rate of arginase formation was produced when these cells, prestarved in presence of proline, were transferred to a “normal” growth medium without added proline. (b) This stimulation of arginase synthesis, brought about by the removal of proline, was not prevented by the addition of actinomycin D and therefore seemed not to be dependent of a formation of new messenger RNA. These results again suggest a proline dependent repression of arginase synthesis at the level of translation of a preformed, stable messenger RNA. Experimental
Cell
Research
48
Repression
of arginase
synthesis
in Chang
liver
17
cells
Thanks are due to Associate Professor Tryggve Gustafson for valuable advice and encouragement. I am also grateful to Professor Harold Strecker for his critical reading of the manuscript. The expert technical assistanceof Miss K. Arvidsson, Miss U. Hammar and Miss E. Nor&r is gratefully acknowledged. This work was supported by grants from the Swedish Cancer Society and from the Swedish National Research Council. REFERENCES
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2-671819
Experimental
Cell Research
48