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Factors affecting acid phosphatase activity in exponential and synchronized L5178Y mouse leukemia cells
Experimental
Cell Research 72 (1972) 465-472
FACTORS AFFECTING ACID PHOSPHATASE ACTIVITY IN EXPONENTIAL AND SYNCHRONIZED L5178Y MOUSE LEUKEMIA CELLS L. N. KAPP’
and S. OKADA
Departments of Experimental Radiology and Radiation Biology and Biophysics, University Rochester School of Medicine and Dentistry, Rochester, N. Y. 14620, USA, and Department of Radiation Biophysics, Faculty of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113 Japan
of
SUMMARY The effects of the following factors on acid phosphatase activity in L517gY cells were studied: inhibitors of RNA, DNA, and protein synthesis, phosphate concentration, growth cycle and cell cycle. Puromycin, high and low phosphate concentrations, and high cell density caused a decrease in enzyme activity while inhibition of DNA synthesis and RNA synthesis caused an increase of activity. During log phase, the cells displayed a constant level of activity which declined to a lower level as the cells entered the stationary phase. In synchronized cells the enzyme activity varied periodically: a minimum in Gl with peaks in late Gl-early S and again in late S-G2. Treatment of cells in the log phase with puromycin caused a linear decay of enzyme activity. In synchronized cells, puromycin also caused a linear decay of enzyme activity at all points in the cell cycle and enzyme activity decayed to levels well below those of controls. From this, it is concluded that the cell regulates its level of acid phosphatase activity by varying the rate of synthesis while the rate of decay remains constant throughout the cell cycle.
In an effort to study regulation of a cell and the progress through its life cycle, a number of enzymes have been studied in several cell lines [l, 2, 7, 8, 12, 15, 16, 171. As a result of these studies, many different patterns of enzyme activity in the cycle have been observed and some attempts at classification and systematization have been made by Mitchison
of enzyme activities in cultured cells. In the present paper, we describe an attempt to study the regulation of activity of acid phosphatase in synchronized L5178Y cells by examining the effects of biological agents and drugs on enzyme activity in log phase and in synchronized cells.
PII. However, at present, there are still no clearly defined schemes or regulatory mechanisms which can account for the variations 1 Present address: Department of Biology, City of Hope National Medical Center, Duarte, Calif. 91010, USA.
MATERIALS
AND METHODS
Cell line Mouse leukemia cells (L5178Y) were used. They were grown in suspension culture in Fischer’s medium supplemented with 10 % horse serum and antibiotics (streptomycin and penicillin). The population doubling time was approx. 9 h. EXP
Cell Res 72
466
L. N. Kapp & S. Okada
Cell counting The cell concentration B Coulter counter.
was determined with a model
Synchrony The synchrony method used was that of Doida & Okada [4]. Cells in early log phase of growth were treated with excess thymidine (2.5 mM) for 5 h. The cells were then centrifuged, the old medium aspirated and fresh warm medium containing Colcemid (0.025 .ng/ml) and deoxycytidine (lO-BM) added immediately. The cells were incubated in this medium for 5 h. At the end of this time, the cells were centrifuged again, the medium aspirated, fresh warm medium added, and the experiment was started.
Drugs Drugs used were Lucanthone (CalBiochem, Los Angeles), Puromycin (Nutritional Biochemicals, Cleveland), Actinomycin D (Mann Research Laboratories, Orangeburg, N. J.), Thymidine (Calbiochem), and 5-bromo-deoxyuridine (Nutritional Biochemicals). Daunomycin was donated by the Meiji Candy Co., Ltd., of Tokyo.
Enzyme assay Acid phosphatase activity was determined by measuring the ability of cell homogenates to convert pnitrophenylphosphate to p-nitrophenol and phosphate [31 Cells were homogenized in a Virtis homogenizer in distilled water. Then an amount of homogenate containing 5 x lo6 cells was added to sodium citrate buffer, 0.05 M, pH 5.0, containing 0.006 M p-nitrophenylphosphate. This was incubated at 37°C for 30 min. To stop the reaction, NaOH was added. The resulting optical density was then read at 410 nm in a Hitachi Spectrophotometer. The cytochrome oxidase assay was performed as described by Wharton [18]. Homogenate containing 5 x lo5 cells was added to potassium phosphate buffer (0.01 M, pH 7) containing reduced cytochrome c. Decrease in optical density was followed at 550 nm. The enzyme activity was proportional to the rate of decrease of the optical density.
Cell fractionation The cell fractionation procedure was that of J. L. Roti Roti [13]. Cells were centrifuged and washed 3 times in a solution containing 0.24 M sucrose, 10 M M&l. 0.01 M Tris-HCl, pH 7. The cell pellet was then frozen immediately, and thawed when needed. After thawing, 1 ml of the buffer was addded to the pellet and the following steps carried out at 0°C: (1) the cell pellet was homogenized for 2 min in an Elvehjem-Potter homogenizer and an additional 1 ml of buffer added; (2) the homogenate was centrifuged at 700 g for 10 min; (3) the supernatant was decanted and the pellet (nuclear fraction) was washed Exptl
Cell Res 72
once with 0.5 ml of buffer and the wash combined with the supernatant; (4) the supematant was centrifuged at 10 000 g for 1 h+ ; (5) the supematant was decanted, the pellet washed once with 5 ml of buffer and recentrifuged. The wash was again combined with the supernatant. This pellet contained the mitochondrial fraction; (6) the resulting supernatant plus wash were spun at 100 000 g for 90 min; (7) the supematant was decanted and pellet washed with 0.5 ml of buffer. The supernatant and the wash were combined to form the soluble fraction, and the final pellet was called the microsomal fraction.
RESULTS Characterization of enzyme activity For acid phosphatase in L5178Y cells, the optical density was found to be linearly proportional to the amount of homogenate used. Similarly, for a given amount of homogenate, the optical density was also found to vary linearly with incubation time. In addition, a pH optimum of approx. 6.0 was also found. From these results, the conditions used for all the following experiments were: 30 min incubation with 5 x lo5 cells. Thus, all results are reported on a basis of activity per cell. Finally, the distribution of acid phosphatase was also determined. Cells were fractionated into nuclear (700 g), mitochondrial (10 000 g), microsomal (100000 g), and soluble fractions. The acid phosphatase activity was measured in each fraction, and Table 1. Enzyme distribution in L.5178Y cells Cells were fractionated as described in Materials and Methods and acid phosphatase and cytochrome oxidase activity was determined in the fractions obtained. Enzyme activity is reported as the percentage of the total activity recovered in each cell fraction
Fraction 700 g pellet, nuclear 10 000 g pellet, mitochondrial (laze oarticulate) 100 *O-g pellet, microsomal (small particulate) 100 000 g supernatant, soluble
Acid phosphatase ( “/I
Cytochrome oxidase (%)
21
2
61
90
1:::
i
Acid phosphatase activity in L5178Y leukemia cells
461
Variation of acid phosphataseduring the growth of L5178Y cells 025
l
l l
0.20150.1 -
0.05 1
1.2x10610" 6x105-
lo5
-
20 30 40 50 60 Fig. 1. Abscissa: age of culture (hours); ordinates: (top) enzyme activity per 5 x lo5 cells in optical density units at 410 nm: (bottom) number of cells/ml as determined by Coulter counter. Enzyme activitv durina L5178Y cell arowth cvcle. Cell concentration was 16 cells/ml at thebeginning of the experiment. As the transition from log to stationary phase occurred, the cell number was counted and the acid phosphatase activity was assayed. 10
simultaneously, as a check on the fractionation procedure, the cytochrome oxidase distribution was also measured. The results are presented in table 1. The bulk of acid phosphatase activity was found in the mitochondrial and lysosomal fractions, but 21% was found in the nuclear fraction. The remaining two fractions displayed some low activity. This could be due to contamination, or possibly a small amount of phosphatase activity actually resided in these fractions. As expected, the cytochrome oxidase activity was found to be mainly (92 “/) in the mitochondrial or large particulate fraction.
Acid phosphatase activity was measured in log phase cells as the cell culture progressed from log to late log and, finally, to stationary phase. These results are shown in fig. 1. During log phase, enzyme activity was constant. As the cells progressed from log to stationary phase, the enzyme activity declined to about 50 or 60 % of its log phase value, and then remained constant in stationary phase at the new low value. In view of these results, all further experiments were carried out at cell concentrations from 2 x loj to 6 x lo5 cells/ml in log phase. Next, the effects of biological factors and of drugs on acid phosphatase activity were tested in log phase cells. These experiments are summarized in table 2. Table 2. Factors which affect acidphosphatase activity in log phase L.5178Y cells The factors tested and their concentrations are listed in the first two columns. These various factors were added to log phase cells and acid phosphatase activity was assayed in control and treated cells after the treatment times listed in the third column. In the fourth column, acid phosphatase activity resulting from the various treatments is reported as percent of controls which were log phase cells. All of these treatments were continuous for the times indicated except for X-irradiation In this case, 10 h after the cells were irradiated, enzyme activity was measured
Factor
Dose
Actinomycin D 0.5 pg/ml Lucanthone 5 w&-n1 Daunomycin 2 Pg/ml Puromycin 20&ml Hydroxyurea 1O-3 M TdR 1O-4 M 1O-5 M BUdR 1O-5 M Phosphate concentration 6 x 1O-3 M -0M X-irradiation 1 000 rads Cell density Stationary phase
Length of treatment (hours)
% of control of activity
3 5 4
130 135 -140 < 40 ,-- 135 -200 100 100
20 20 10
22
60
1:; 55 Exptl Cell Res 72
468
L. N. Kapp & S. Okada s
/M/G,/
/G2
taken and assayed for acid phosphatase activity. These results are shown in fig. 2. Acid phosphatase activity in L5178Y cells. seems to have two peaks: one small and somewhat variable peak in late Gl-early S, and a larger major peak in late S.
/
0.25
Effects of hydroxyurea
3
4
5
6
7
8
9
10
Fig. 2. Abscissa: time (hours) following release from Colcemid; ordinates: (top) enzyme activity per 5 x lo5 cells in optical density units in nm; (bottom) cells/ml. Enzyme activity in synchronized cells. Cells were synchronized by the method of Doida & Okada. After release from Colcemid at 0 time, samples were removed and assayed for acid phosphatase activity.
Inhibitors of RNA synthesis caused an increase in acid phosphatase activity (this effect will be discussed in a subsequent paper). Inhibition of DNA synthesis by hydroxyurea and thymidine also caused an increase in activity. Thymidine at low concentration and bromodeoxyuridine had no effects. Factors which reduced enzyme activity were (1) inhibition of protein synthesis by addition of puromycin; (2) high and low phosphate concentrations; (3) high cell densities (stationary phase growth conditions). Enzyme
activity
in synchronized
PIEffects of puromycin on acid phosphatase activity
Puromycin (20 pg/ml) was added to log phase and synchronized cultures and the decay of enzyme activity was followed. Fig. 4 shows the effects of puromycin added at zero-time to log phase cells. Although puromycin at this concentration
cells
Next, the enzyme activity was measured during the cell cycle in synchronized cells. The cells were synchronized by the method of Doida & Okada utilizing an excess thymidine treatment followed by a Colcemid treatment. Upon release from Colcemid, samples were Exptl Cell Res 72
The effects of inhibition of DNA synthesis and of protein synthesis on acid phosphatase activity were studied in more detail. Fig. 3 shows the time course of enzyme activity following the addition of hydroxyurea to the cells. There was no apparent effect for 3 h. After this, the enzyme activity began to rise and continued to do so until at least the fifth hour after addition of the drug. Induction of thymidine kinase in Chang liver cells by hydroxyurea has also been reported by Eker
Fig. 3. Abscissa: time after addition of hydroxyurea (hours); ordinate: enzyme activity per 5 x IO5 cells as percent of control. Effect of hydroxyurea (IO-*M) on enzyme activity. Cells were treated with hydroxyurea at time zero. At intervals samples were removed from treated and control cultures and assayed for acid phosphatase activity.
Acid phosphatase activity in L5178 Y leukemia cells
469
Table 3. Effect of puromycin on acid phosphatase activity in synchronized cells Cells were synchronized as described in the text. The synchronized cells were then divided into two fractions, one of which was treated with puromycin (20 ,ug/ml) and the other served as the control. Decay of acid phosphatase was followed for 2 to 3 h after the addition of puromycin. The times in the cell cycle when puromycin was added is shown, as well as the presence or absence of a lag phase before decay of enzyme activity, and the half-life of the enzyme activity. ‘Half-lives’ were determined from the slopes (on linear plots) of curves fitted to the data down to about 40 % residual activity 0'
20
40
60'
do
loo
120
Fig. 4. Abscissa: time after addition of puromycin (min); ordinate: enzyme activity per 5 x lo5 cells as percentage of control. Effect of puromycin on enzyme activity. Puromycin (20 pg/ml) was added to cells at time zero. Samples of treated and control cultures were removed at intervals and assayed for acid phosphatase activity. Results are reported as percentage of control as described in fig. 4.
0.4 w
0.25 7
O.'O i
/M/G,/
0.101
I
I
2.8 3.0 3.3 4.2 4.2 5.0
Stage of cell cycle
Lag before decay of activity (min)
Gl-S boundary Gl-S boundary S S S S
Half-life of enzyme activity (min)
0
220
0
155
30 0 0 50
165 260
180 195
Mean half-life = 196 f 38 min.
s
I
Time of treatment after release from Colcemid (hours)
I
I
/G*+M
I
I
I
I
,
012345 4 7 a 9 10 Fig. 5. Abscissa: time after release from Colcemid (hours); ordinates: enzyme activity per 5 x lo5 cells in optical density units. Effect of puromycin on enzyme activity in synchronized cells. Cells were synchronized as described in the text, and divided into two cultures. One was treated wrth puromycin (20 yg/ml) and the other served as control. Enzyme activity was followed in treated and control synchonized populations. 0, Control cells; l , treated cells. Top: Puromycin was added at mtd-S during declining enzyme activity. Bottom: Puromycin was added in the latter half of S while enzyme activity wasIrking.
inhibits incorporation of 3H-leucine by 80% in the first 30 min [5], no effect was observable on acid phosphatase activity for 40 min. Thereafter, the activity decayed with apparent first order kinetics for 2 h. The cell number showed little change over this same period. Next, the effect of puromycin was observed in synchronized cells. The drug was added during each rise or decline of enzyme activity shown in fig. 2, and the decay of activity was followed for 3 h thereafter. The plateau shown in fig. 4 for log phase cells was not found in all parts of the cell cycle; during declining periods of acid phosphatase activity, no plateau phase occurred. However, during periods of increasing enzyme activity, plateaus were found. Fig. 5 shows the results when puromycin was added at several points during S phase in the cell cycle. Exptl Cell Res 72
470
L. N. Kapp & S. Okada
The upper panel of fig. 5 shows the effects of puromycin when added during a period of declining enzyme activity. No plateaus were visible. The bottom part of fig. 5 shows the results when the drug was added while the enzyme activity was rising. In this case, a plateau was visible in the treated cells. Finally, table 3 summarizes the results of the puromycin experiments over the cell cycle. In all cases, acid phosphatase activity decayed for 3 h and to levels well below those of the controls. In addition, the average half life was 196& 38 min. DISCUSSION Under normal conditions, log phase cells display a constant level of acid phosphatase activity (fig. 2). In order to gain some insight into the regulatory mechanisms by which the cell controls its enzyme activity, the effects of various biological factors and drugs on acid phosphatase activity were examined. Several biological factors were studied, including stationary phase vs log phase cells, phosphate concentrations in the medium, and cell cycle behaviour during one cell cycle. When the cells’ progress from log phase to stationary phase occurred, acid phosphatase activity began to decrease several hours before the cell number reached the stationary phase. The enzyme activity continued to decrease and remained at about half of that of the log phase cells. When the cells were placed in high phosphate medium or in phosphate-free medium, little or no increase in cell number was observed. The enzyme activity in both situations was about one quarter of that of the log phase cells in the normal phosphate-containing medium (normal medium contained approx 1 mM phosphate concentration). It is still not clear whether low enzyme activity in high and very low, or zero, concentrations of phosphate is Exptl
Cell
Res 72
a phenomena similar to that of low enzyme activity observed in stationary phase cells. Upon addition of puromycin to log phase cells, there was a 40 min latent period before enzyme activity began to decay. Perhaps this may be the time necessary for some kind of ‘assembly step’, such as the time to reach final tertiary or quaternary structure, or perhaps, for the ‘packaging’ of the enzymes in lysosomes or some other final working site. This could account for the 40 min latent period, whereas decay of activity represents breakdown of the enzyme. Although the data are not shown here, it was found that addition of cycloheximide to log phase cells at concentrations from 1 ,ug/ ml up to 100 pg/ml had no effect on acid phosphatase activity. Exposures of 3 or 4 h to the drug produced no measurable decline of enzyme activity, in spite of the fact that cycloheximide at such concentrations inhibited 3H-leucine incorporation at least as effectively as puromycin did. At present, no explanation can be offered for such differences in cellular response to the two drugs. Three inhibitors of RNA synthesis (actinomycin D, lucanthone and daunomycin) caused an increase of acid phosphatase activity. Possibly this increase results from the decay of a short-lived repressor-RNA and inhibition of its synthesis as proposed by Tomkins et al. [16]. A further detailed discussion of this will be given in a subsequent paper. DNA synthesis inhibitors (i.e., hydroxyurea and excess thymidine) and X-irradiation also produced an increase of acid phosphatase activity. Although thymidine and hydroxyurea inhibit DNA synthesis immediately, the increase of acid phosphatase is a slow gradual process. Thus, it seems that inhibition of DNA synthesis per se is not directly coupled with regulation of the enzyme level, but that other processes resulting from inhibition of DNA synthesis, e.g., accumulation of the
Acid phosphatase activity in L5178Y leukemia cells
cells at S stage, might be a contributing factor, since acid phosphatase levels vary during the cell cycle and display a maximum activity during S phase. It is interesting to note that incorporation of bromodeoxyuridine into cellular DNA is said to inhibit normal levels and induction of tyrosine amino transferase by hormones or actinomycin D [14] in hepatoma tissue culture cells. However, for actinomycin D-inducible acid phosphatase of L5178Y cells, bromodeoxyuridine incorporation showed no effect on enzyme levels; that BUdR is incorporated under these conditions has already been shown [12a]. Another biological factor examined was the cell cycle. Previous work of Bosmann et al. [I] failed to disclose any cyclic change of acid phosphatase activity in L5178Y cells. In contrast, the present experiment shows cyclic variations of phosphatase activity in the cell cycle; one minor and somewhat variable increase in the Gl-early S stage and the second major increase in the middle-late S stage. It was found that small, but significant cyclic variations of acid phosphatase could be obliterated easily when averaging several imperfectly synchronized populations. Bosmann’s failure (with L5178Y cells) to observe cyclic variation of this enzyme might be attributed to this. Churchill 8z Studzinski [2] found that acid phosphatase showed a pattern of activity in HeLa cells somewhat similar to that reported here except for the absence of the smaller first peak. Examples of cyclic variation of other enzymes in other cell lines are glucose-6-phosphate dehydrogenase and lactate dehydrogenase in Chinese hamster cells [7, 81, thymidine kinase in mouse L cells [9] ribonucleotide reductase in L cells [ 11, and thymidine kinase in Chinese hamster cells [15]. In addition, Klevecz [7] and Klevecz & Ruddle [8] found that lactate dehydrogenase displayed several peaks during the cell cycle, similar to the behavior of acid
471
phosphatase reported here. So far, however, no common characteristics have been found among these enzymes. In order to gain more information on cell regulatory mechanisms, the enzyme activity was followed in synchronized cells for one generation time and the puromycin studies were carried out in more detail in log phase and in synchronized cells. Regardless of when in the cell cycle puromycin was added, the enzyme activity decayed to well below the levels of the control with a half-life of 196 min. This compares with half-lives of 2 h for lactate dehydrogenase in Chinese hamster cells as found by Klevecz [7] and of 4 to 5 h for tyrosine aminotransferase in hepatoma tissue culture cells as reported by Martin et al. [lo]. In addition, the latent phases before decay of enzyme activity were only observed in late Gl-early S and in late S during periods when enzyme activity was rising. This suggests the following: (1) Acid phosphatase is synthesized throughout the cell cycle and has a rate of decay which is independent of cell cycle position. (2) The peaks of enzyme activity at G learly S and in late S are due to higher rates of enzyme synthesis than at other parts of the cell cycle. Thus, it would seem that the cell regulates its acid phosphatase activity during the cell cycle primarily by altering its rate of synthesis of new protein. Similar conclusions were also reached by Tomkins et al., for tyrosine aminotransferase in hepatoma tissue culture cells [16] and by Klevecz for lactate dehydrogenase in Chinese hamster cells [7]. They confirmed the actual existence of turnover by immunological precipitation of radioactive enzyme protein. They found that the loss of activity, as measured by biochemical assays, paralleled the loss of incorporated amino acids as measured by the loss of radioactivity in immunoprecipitated proteins. Exptl
Cell Res 72
472
L. Kapp & S. Okada
Transcriptional events clearly play some role in regulation of protein synthesis; however, to understand more about this, additional and more detailed experiments would have to be carried out involving RNA synthesis inhibitors in log phase and in synchronized cells. Such experiments will be reported in a subsequent paper. This work was supported, partially, by a US Atomic Energy Commission Contract (AT(30-l)1286), by a contract with the US Atomic Energy Commission at the University of Rochester Atomic Energy Project (UR-3409-8), and by a Cancer grant from the Ministry of Education in Japan.
REFERENCES 1. Bosmann, H B & Bernacki, R J, Exptl cell res 61 (1970) 379. 2. Churchill, J R & Studzinski, G P, J cell physiol 75 (1970) 297. 3. Christofalo, U J, Kabakjian, J R & Kritchevski, D, Proc sot exptl biol med 126 (1967) 273. 4. Doida, Y & Okada, S, ?xptl cell res 48 (1967) 540.
Exptl
Cell Res 72
-
Cell tissue kinet 5 (1972) 15.
2. Eker, P, J biol them 243 (1968) 1979. 7: Klevecz, R R, J cell bio143 (1969) 207. 8. Klevecz, R R & Ruddle, F H, Science 159 (1968) 634. 9. Littlefield, J W, Biochim biophys acta 114 (1966) 1. 398. Martin, D, Tomkins, G M & Bressler, M, Proc ’ natl acad sci US 63 (1969) 842. 11. Mitchison, J M, Science 165 (1969) 657.
12. Murphree, S, Stubblefield, E & Moore, E C, Exptl cell res 58 (1969) 118. 12a. Okada, S, Biophys j 8 (1968) 650. 13. Roti Roti, 3 L & Okada, S, Biophys sot abstracts 11 (1971) 263a. 14. Stellwagen, A & Tomkins, G M, J mol biol 56 (1971) 167. 1.5. Stubblefield, E & Murphree, S, Exptl ccl1 res 48 (1967) 652. 16. Gelehrter, T D, Grunner, D, Martin, D Jr, Samuels, H H & Thompson, E B, Science 166 (1969) 1474. 17. Turner, M K, Abrams, R & Leiberman, I, J biol them 243 (1968) 3725. 18. Wharton, D C & Tzagoloff, A, Methods in enzymology (ed R Estabrook & M Pullman) vol. 10, p. 245. Academic Press, New York (1967).
Received September 21, 1971 Revised version received January 10, 1972
Cell Research 72 (1972) 465-472
FACTORS AFFECTING ACID PHOSPHATASE ACTIVITY IN EXPONENTIAL AND SYNCHRONIZED L5178Y MOUSE LEUKEMIA CELLS L. N. KAPP’
and S. OKADA
Departments of Experimental Radiology and Radiation Biology and Biophysics, University Rochester School of Medicine and Dentistry, Rochester, N. Y. 14620, USA, and Department of Radiation Biophysics, Faculty of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113 Japan
of
SUMMARY The effects of the following factors on acid phosphatase activity in L517gY cells were studied: inhibitors of RNA, DNA, and protein synthesis, phosphate concentration, growth cycle and cell cycle. Puromycin, high and low phosphate concentrations, and high cell density caused a decrease in enzyme activity while inhibition of DNA synthesis and RNA synthesis caused an increase of activity. During log phase, the cells displayed a constant level of activity which declined to a lower level as the cells entered the stationary phase. In synchronized cells the enzyme activity varied periodically: a minimum in Gl with peaks in late Gl-early S and again in late S-G2. Treatment of cells in the log phase with puromycin caused a linear decay of enzyme activity. In synchronized cells, puromycin also caused a linear decay of enzyme activity at all points in the cell cycle and enzyme activity decayed to levels well below those of controls. From this, it is concluded that the cell regulates its level of acid phosphatase activity by varying the rate of synthesis while the rate of decay remains constant throughout the cell cycle.
In an effort to study regulation of a cell and the progress through its life cycle, a number of enzymes have been studied in several cell lines [l, 2, 7, 8, 12, 15, 16, 171. As a result of these studies, many different patterns of enzyme activity in the cycle have been observed and some attempts at classification and systematization have been made by Mitchison
of enzyme activities in cultured cells. In the present paper, we describe an attempt to study the regulation of activity of acid phosphatase in synchronized L5178Y cells by examining the effects of biological agents and drugs on enzyme activity in log phase and in synchronized cells.
PII. However, at present, there are still no clearly defined schemes or regulatory mechanisms which can account for the variations 1 Present address: Department of Biology, City of Hope National Medical Center, Duarte, Calif. 91010, USA.
MATERIALS
AND METHODS
Cell line Mouse leukemia cells (L5178Y) were used. They were grown in suspension culture in Fischer’s medium supplemented with 10 % horse serum and antibiotics (streptomycin and penicillin). The population doubling time was approx. 9 h. EXP
Cell Res 72
466
L. N. Kapp & S. Okada
Cell counting The cell concentration B Coulter counter.
was determined with a model
Synchrony The synchrony method used was that of Doida & Okada [4]. Cells in early log phase of growth were treated with excess thymidine (2.5 mM) for 5 h. The cells were then centrifuged, the old medium aspirated and fresh warm medium containing Colcemid (0.025 .ng/ml) and deoxycytidine (lO-BM) added immediately. The cells were incubated in this medium for 5 h. At the end of this time, the cells were centrifuged again, the medium aspirated, fresh warm medium added, and the experiment was started.
Drugs Drugs used were Lucanthone (CalBiochem, Los Angeles), Puromycin (Nutritional Biochemicals, Cleveland), Actinomycin D (Mann Research Laboratories, Orangeburg, N. J.), Thymidine (Calbiochem), and 5-bromo-deoxyuridine (Nutritional Biochemicals). Daunomycin was donated by the Meiji Candy Co., Ltd., of Tokyo.
Enzyme assay Acid phosphatase activity was determined by measuring the ability of cell homogenates to convert pnitrophenylphosphate to p-nitrophenol and phosphate [31 Cells were homogenized in a Virtis homogenizer in distilled water. Then an amount of homogenate containing 5 x lo6 cells was added to sodium citrate buffer, 0.05 M, pH 5.0, containing 0.006 M p-nitrophenylphosphate. This was incubated at 37°C for 30 min. To stop the reaction, NaOH was added. The resulting optical density was then read at 410 nm in a Hitachi Spectrophotometer. The cytochrome oxidase assay was performed as described by Wharton [18]. Homogenate containing 5 x lo5 cells was added to potassium phosphate buffer (0.01 M, pH 7) containing reduced cytochrome c. Decrease in optical density was followed at 550 nm. The enzyme activity was proportional to the rate of decrease of the optical density.
Cell fractionation The cell fractionation procedure was that of J. L. Roti Roti [13]. Cells were centrifuged and washed 3 times in a solution containing 0.24 M sucrose, 10 M M&l. 0.01 M Tris-HCl, pH 7. The cell pellet was then frozen immediately, and thawed when needed. After thawing, 1 ml of the buffer was addded to the pellet and the following steps carried out at 0°C: (1) the cell pellet was homogenized for 2 min in an Elvehjem-Potter homogenizer and an additional 1 ml of buffer added; (2) the homogenate was centrifuged at 700 g for 10 min; (3) the supernatant was decanted and the pellet (nuclear fraction) was washed Exptl
Cell Res 72
once with 0.5 ml of buffer and the wash combined with the supernatant; (4) the supematant was centrifuged at 10 000 g for 1 h+ ; (5) the supematant was decanted, the pellet washed once with 5 ml of buffer and recentrifuged. The wash was again combined with the supernatant. This pellet contained the mitochondrial fraction; (6) the resulting supernatant plus wash were spun at 100 000 g for 90 min; (7) the supematant was decanted and pellet washed with 0.5 ml of buffer. The supernatant and the wash were combined to form the soluble fraction, and the final pellet was called the microsomal fraction.
RESULTS Characterization of enzyme activity For acid phosphatase in L5178Y cells, the optical density was found to be linearly proportional to the amount of homogenate used. Similarly, for a given amount of homogenate, the optical density was also found to vary linearly with incubation time. In addition, a pH optimum of approx. 6.0 was also found. From these results, the conditions used for all the following experiments were: 30 min incubation with 5 x lo5 cells. Thus, all results are reported on a basis of activity per cell. Finally, the distribution of acid phosphatase was also determined. Cells were fractionated into nuclear (700 g), mitochondrial (10 000 g), microsomal (100000 g), and soluble fractions. The acid phosphatase activity was measured in each fraction, and Table 1. Enzyme distribution in L.5178Y cells Cells were fractionated as described in Materials and Methods and acid phosphatase and cytochrome oxidase activity was determined in the fractions obtained. Enzyme activity is reported as the percentage of the total activity recovered in each cell fraction
Fraction 700 g pellet, nuclear 10 000 g pellet, mitochondrial (laze oarticulate) 100 *O-g pellet, microsomal (small particulate) 100 000 g supernatant, soluble
Acid phosphatase ( “/I
Cytochrome oxidase (%)
21
2
61
90
1:::
i
Acid phosphatase activity in L5178Y leukemia cells
461
Variation of acid phosphataseduring the growth of L5178Y cells 025
l
l l
0.20150.1 -
0.05 1
1.2x10610" 6x105-
lo5
-
20 30 40 50 60 Fig. 1. Abscissa: age of culture (hours); ordinates: (top) enzyme activity per 5 x lo5 cells in optical density units at 410 nm: (bottom) number of cells/ml as determined by Coulter counter. Enzyme activitv durina L5178Y cell arowth cvcle. Cell concentration was 16 cells/ml at thebeginning of the experiment. As the transition from log to stationary phase occurred, the cell number was counted and the acid phosphatase activity was assayed. 10
simultaneously, as a check on the fractionation procedure, the cytochrome oxidase distribution was also measured. The results are presented in table 1. The bulk of acid phosphatase activity was found in the mitochondrial and lysosomal fractions, but 21% was found in the nuclear fraction. The remaining two fractions displayed some low activity. This could be due to contamination, or possibly a small amount of phosphatase activity actually resided in these fractions. As expected, the cytochrome oxidase activity was found to be mainly (92 “/) in the mitochondrial or large particulate fraction.
Acid phosphatase activity was measured in log phase cells as the cell culture progressed from log to late log and, finally, to stationary phase. These results are shown in fig. 1. During log phase, enzyme activity was constant. As the cells progressed from log to stationary phase, the enzyme activity declined to about 50 or 60 % of its log phase value, and then remained constant in stationary phase at the new low value. In view of these results, all further experiments were carried out at cell concentrations from 2 x loj to 6 x lo5 cells/ml in log phase. Next, the effects of biological factors and of drugs on acid phosphatase activity were tested in log phase cells. These experiments are summarized in table 2. Table 2. Factors which affect acidphosphatase activity in log phase L.5178Y cells The factors tested and their concentrations are listed in the first two columns. These various factors were added to log phase cells and acid phosphatase activity was assayed in control and treated cells after the treatment times listed in the third column. In the fourth column, acid phosphatase activity resulting from the various treatments is reported as percent of controls which were log phase cells. All of these treatments were continuous for the times indicated except for X-irradiation In this case, 10 h after the cells were irradiated, enzyme activity was measured
Factor
Dose
Actinomycin D 0.5 pg/ml Lucanthone 5 w&-n1 Daunomycin 2 Pg/ml Puromycin 20&ml Hydroxyurea 1O-3 M TdR 1O-4 M 1O-5 M BUdR 1O-5 M Phosphate concentration 6 x 1O-3 M -0M X-irradiation 1 000 rads Cell density Stationary phase
Length of treatment (hours)
% of control of activity
3 5 4
130 135 -140 < 40 ,-- 135 -200 100 100
20 20 10
22
60
1:; 55 Exptl Cell Res 72
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L. N. Kapp & S. Okada s
/M/G,/
/G2
taken and assayed for acid phosphatase activity. These results are shown in fig. 2. Acid phosphatase activity in L5178Y cells. seems to have two peaks: one small and somewhat variable peak in late Gl-early S, and a larger major peak in late S.
/
0.25
Effects of hydroxyurea
3
4
5
6
7
8
9
10
Fig. 2. Abscissa: time (hours) following release from Colcemid; ordinates: (top) enzyme activity per 5 x lo5 cells in optical density units in nm; (bottom) cells/ml. Enzyme activity in synchronized cells. Cells were synchronized by the method of Doida & Okada. After release from Colcemid at 0 time, samples were removed and assayed for acid phosphatase activity.
Inhibitors of RNA synthesis caused an increase in acid phosphatase activity (this effect will be discussed in a subsequent paper). Inhibition of DNA synthesis by hydroxyurea and thymidine also caused an increase in activity. Thymidine at low concentration and bromodeoxyuridine had no effects. Factors which reduced enzyme activity were (1) inhibition of protein synthesis by addition of puromycin; (2) high and low phosphate concentrations; (3) high cell densities (stationary phase growth conditions). Enzyme
activity
in synchronized
PIEffects of puromycin on acid phosphatase activity
Puromycin (20 pg/ml) was added to log phase and synchronized cultures and the decay of enzyme activity was followed. Fig. 4 shows the effects of puromycin added at zero-time to log phase cells. Although puromycin at this concentration
cells
Next, the enzyme activity was measured during the cell cycle in synchronized cells. The cells were synchronized by the method of Doida & Okada utilizing an excess thymidine treatment followed by a Colcemid treatment. Upon release from Colcemid, samples were Exptl Cell Res 72
The effects of inhibition of DNA synthesis and of protein synthesis on acid phosphatase activity were studied in more detail. Fig. 3 shows the time course of enzyme activity following the addition of hydroxyurea to the cells. There was no apparent effect for 3 h. After this, the enzyme activity began to rise and continued to do so until at least the fifth hour after addition of the drug. Induction of thymidine kinase in Chang liver cells by hydroxyurea has also been reported by Eker
Fig. 3. Abscissa: time after addition of hydroxyurea (hours); ordinate: enzyme activity per 5 x IO5 cells as percent of control. Effect of hydroxyurea (IO-*M) on enzyme activity. Cells were treated with hydroxyurea at time zero. At intervals samples were removed from treated and control cultures and assayed for acid phosphatase activity.
Acid phosphatase activity in L5178 Y leukemia cells
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Table 3. Effect of puromycin on acid phosphatase activity in synchronized cells Cells were synchronized as described in the text. The synchronized cells were then divided into two fractions, one of which was treated with puromycin (20 ,ug/ml) and the other served as the control. Decay of acid phosphatase was followed for 2 to 3 h after the addition of puromycin. The times in the cell cycle when puromycin was added is shown, as well as the presence or absence of a lag phase before decay of enzyme activity, and the half-life of the enzyme activity. ‘Half-lives’ were determined from the slopes (on linear plots) of curves fitted to the data down to about 40 % residual activity 0'
20
40
60'
do
loo
120
Fig. 4. Abscissa: time after addition of puromycin (min); ordinate: enzyme activity per 5 x lo5 cells as percentage of control. Effect of puromycin on enzyme activity. Puromycin (20 pg/ml) was added to cells at time zero. Samples of treated and control cultures were removed at intervals and assayed for acid phosphatase activity. Results are reported as percentage of control as described in fig. 4.
0.4 w
0.25 7
O.'O i
/M/G,/
0.101
I
I
2.8 3.0 3.3 4.2 4.2 5.0
Stage of cell cycle
Lag before decay of activity (min)
Gl-S boundary Gl-S boundary S S S S
Half-life of enzyme activity (min)
0
220
0
155
30 0 0 50
165 260
180 195
Mean half-life = 196 f 38 min.
s
I
Time of treatment after release from Colcemid (hours)
I
I
/G*+M
I
I
I
I
,
012345 4 7 a 9 10 Fig. 5. Abscissa: time after release from Colcemid (hours); ordinates: enzyme activity per 5 x lo5 cells in optical density units. Effect of puromycin on enzyme activity in synchronized cells. Cells were synchronized as described in the text, and divided into two cultures. One was treated wrth puromycin (20 yg/ml) and the other served as control. Enzyme activity was followed in treated and control synchonized populations. 0, Control cells; l , treated cells. Top: Puromycin was added at mtd-S during declining enzyme activity. Bottom: Puromycin was added in the latter half of S while enzyme activity wasIrking.
inhibits incorporation of 3H-leucine by 80% in the first 30 min [5], no effect was observable on acid phosphatase activity for 40 min. Thereafter, the activity decayed with apparent first order kinetics for 2 h. The cell number showed little change over this same period. Next, the effect of puromycin was observed in synchronized cells. The drug was added during each rise or decline of enzyme activity shown in fig. 2, and the decay of activity was followed for 3 h thereafter. The plateau shown in fig. 4 for log phase cells was not found in all parts of the cell cycle; during declining periods of acid phosphatase activity, no plateau phase occurred. However, during periods of increasing enzyme activity, plateaus were found. Fig. 5 shows the results when puromycin was added at several points during S phase in the cell cycle. Exptl Cell Res 72
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L. N. Kapp & S. Okada
The upper panel of fig. 5 shows the effects of puromycin when added during a period of declining enzyme activity. No plateaus were visible. The bottom part of fig. 5 shows the results when the drug was added while the enzyme activity was rising. In this case, a plateau was visible in the treated cells. Finally, table 3 summarizes the results of the puromycin experiments over the cell cycle. In all cases, acid phosphatase activity decayed for 3 h and to levels well below those of the controls. In addition, the average half life was 196& 38 min. DISCUSSION Under normal conditions, log phase cells display a constant level of acid phosphatase activity (fig. 2). In order to gain some insight into the regulatory mechanisms by which the cell controls its enzyme activity, the effects of various biological factors and drugs on acid phosphatase activity were examined. Several biological factors were studied, including stationary phase vs log phase cells, phosphate concentrations in the medium, and cell cycle behaviour during one cell cycle. When the cells’ progress from log phase to stationary phase occurred, acid phosphatase activity began to decrease several hours before the cell number reached the stationary phase. The enzyme activity continued to decrease and remained at about half of that of the log phase cells. When the cells were placed in high phosphate medium or in phosphate-free medium, little or no increase in cell number was observed. The enzyme activity in both situations was about one quarter of that of the log phase cells in the normal phosphate-containing medium (normal medium contained approx 1 mM phosphate concentration). It is still not clear whether low enzyme activity in high and very low, or zero, concentrations of phosphate is Exptl
Cell
Res 72
a phenomena similar to that of low enzyme activity observed in stationary phase cells. Upon addition of puromycin to log phase cells, there was a 40 min latent period before enzyme activity began to decay. Perhaps this may be the time necessary for some kind of ‘assembly step’, such as the time to reach final tertiary or quaternary structure, or perhaps, for the ‘packaging’ of the enzymes in lysosomes or some other final working site. This could account for the 40 min latent period, whereas decay of activity represents breakdown of the enzyme. Although the data are not shown here, it was found that addition of cycloheximide to log phase cells at concentrations from 1 ,ug/ ml up to 100 pg/ml had no effect on acid phosphatase activity. Exposures of 3 or 4 h to the drug produced no measurable decline of enzyme activity, in spite of the fact that cycloheximide at such concentrations inhibited 3H-leucine incorporation at least as effectively as puromycin did. At present, no explanation can be offered for such differences in cellular response to the two drugs. Three inhibitors of RNA synthesis (actinomycin D, lucanthone and daunomycin) caused an increase of acid phosphatase activity. Possibly this increase results from the decay of a short-lived repressor-RNA and inhibition of its synthesis as proposed by Tomkins et al. [16]. A further detailed discussion of this will be given in a subsequent paper. DNA synthesis inhibitors (i.e., hydroxyurea and excess thymidine) and X-irradiation also produced an increase of acid phosphatase activity. Although thymidine and hydroxyurea inhibit DNA synthesis immediately, the increase of acid phosphatase is a slow gradual process. Thus, it seems that inhibition of DNA synthesis per se is not directly coupled with regulation of the enzyme level, but that other processes resulting from inhibition of DNA synthesis, e.g., accumulation of the
Acid phosphatase activity in L5178Y leukemia cells
cells at S stage, might be a contributing factor, since acid phosphatase levels vary during the cell cycle and display a maximum activity during S phase. It is interesting to note that incorporation of bromodeoxyuridine into cellular DNA is said to inhibit normal levels and induction of tyrosine amino transferase by hormones or actinomycin D [14] in hepatoma tissue culture cells. However, for actinomycin D-inducible acid phosphatase of L5178Y cells, bromodeoxyuridine incorporation showed no effect on enzyme levels; that BUdR is incorporated under these conditions has already been shown [12a]. Another biological factor examined was the cell cycle. Previous work of Bosmann et al. [I] failed to disclose any cyclic change of acid phosphatase activity in L5178Y cells. In contrast, the present experiment shows cyclic variations of phosphatase activity in the cell cycle; one minor and somewhat variable increase in the Gl-early S stage and the second major increase in the middle-late S stage. It was found that small, but significant cyclic variations of acid phosphatase could be obliterated easily when averaging several imperfectly synchronized populations. Bosmann’s failure (with L5178Y cells) to observe cyclic variation of this enzyme might be attributed to this. Churchill 8z Studzinski [2] found that acid phosphatase showed a pattern of activity in HeLa cells somewhat similar to that reported here except for the absence of the smaller first peak. Examples of cyclic variation of other enzymes in other cell lines are glucose-6-phosphate dehydrogenase and lactate dehydrogenase in Chinese hamster cells [7, 81, thymidine kinase in mouse L cells [9] ribonucleotide reductase in L cells [ 11, and thymidine kinase in Chinese hamster cells [15]. In addition, Klevecz [7] and Klevecz & Ruddle [8] found that lactate dehydrogenase displayed several peaks during the cell cycle, similar to the behavior of acid
471
phosphatase reported here. So far, however, no common characteristics have been found among these enzymes. In order to gain more information on cell regulatory mechanisms, the enzyme activity was followed in synchronized cells for one generation time and the puromycin studies were carried out in more detail in log phase and in synchronized cells. Regardless of when in the cell cycle puromycin was added, the enzyme activity decayed to well below the levels of the control with a half-life of 196 min. This compares with half-lives of 2 h for lactate dehydrogenase in Chinese hamster cells as found by Klevecz [7] and of 4 to 5 h for tyrosine aminotransferase in hepatoma tissue culture cells as reported by Martin et al. [lo]. In addition, the latent phases before decay of enzyme activity were only observed in late Gl-early S and in late S during periods when enzyme activity was rising. This suggests the following: (1) Acid phosphatase is synthesized throughout the cell cycle and has a rate of decay which is independent of cell cycle position. (2) The peaks of enzyme activity at G learly S and in late S are due to higher rates of enzyme synthesis than at other parts of the cell cycle. Thus, it would seem that the cell regulates its acid phosphatase activity during the cell cycle primarily by altering its rate of synthesis of new protein. Similar conclusions were also reached by Tomkins et al., for tyrosine aminotransferase in hepatoma tissue culture cells [16] and by Klevecz for lactate dehydrogenase in Chinese hamster cells [7]. They confirmed the actual existence of turnover by immunological precipitation of radioactive enzyme protein. They found that the loss of activity, as measured by biochemical assays, paralleled the loss of incorporated amino acids as measured by the loss of radioactivity in immunoprecipitated proteins. Exptl
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L. Kapp & S. Okada
Transcriptional events clearly play some role in regulation of protein synthesis; however, to understand more about this, additional and more detailed experiments would have to be carried out involving RNA synthesis inhibitors in log phase and in synchronized cells. Such experiments will be reported in a subsequent paper. This work was supported, partially, by a US Atomic Energy Commission Contract (AT(30-l)1286), by a contract with the US Atomic Energy Commission at the University of Rochester Atomic Energy Project (UR-3409-8), and by a Cancer grant from the Ministry of Education in Japan.
REFERENCES 1. Bosmann, H B & Bernacki, R J, Exptl cell res 61 (1970) 379. 2. Churchill, J R & Studzinski, G P, J cell physiol 75 (1970) 297. 3. Christofalo, U J, Kabakjian, J R & Kritchevski, D, Proc sot exptl biol med 126 (1967) 273. 4. Doida, Y & Okada, S, ?xptl cell res 48 (1967) 540.
Exptl
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Cell tissue kinet 5 (1972) 15.
2. Eker, P, J biol them 243 (1968) 1979. 7: Klevecz, R R, J cell bio143 (1969) 207. 8. Klevecz, R R & Ruddle, F H, Science 159 (1968) 634. 9. Littlefield, J W, Biochim biophys acta 114 (1966) 1. 398. Martin, D, Tomkins, G M & Bressler, M, Proc ’ natl acad sci US 63 (1969) 842. 11. Mitchison, J M, Science 165 (1969) 657.
12. Murphree, S, Stubblefield, E & Moore, E C, Exptl cell res 58 (1969) 118. 12a. Okada, S, Biophys j 8 (1968) 650. 13. Roti Roti, 3 L & Okada, S, Biophys sot abstracts 11 (1971) 263a. 14. Stellwagen, A & Tomkins, G M, J mol biol 56 (1971) 167. 1.5. Stubblefield, E & Murphree, S, Exptl ccl1 res 48 (1967) 652. 16. Gelehrter, T D, Grunner, D, Martin, D Jr, Samuels, H H & Thompson, E B, Science 166 (1969) 1474. 17. Turner, M K, Abrams, R & Leiberman, I, J biol them 243 (1968) 3725. 18. Wharton, D C & Tzagoloff, A, Methods in enzymology (ed R Estabrook & M Pullman) vol. 10, p. 245. Academic Press, New York (1967).
Received September 21, 1971 Revised version received January 10, 1972