DNA polymerase activity in phytohemagglutinin-stimulated and non-stimulated human lymphocytes

Copyright All rights

0 1973 by Academic Press, Inc. of reproduction in any form rexrued

Experimental

Cell Research 77 (1973) 415-421

DNA POLYMERASE ACTIVITY IN PHYTOHEMAGGLUTININSTIMULATED AND NON-STIMULATED HUMAN LYMPHOCYTES GERDA

TYRSTED,

Biochemical

Institute

BIRGITTE B, University

MUNCH-PETERSEN of Copenhagen,

DK-2100

and LISBET CLOOS Copenhagen,

Denmark

SUMMARY Requirements and optimal conditions have been studied for the activity of DNA oolvmerase from phytohemagglutinin-stimulated and non-stimulated human lymphocytes. Differences were found in thermal stabilitv and inhibitory effect of KC1 and n-chloromercuribenzoate. The relationship was determined between DNA polymerase activity, cellular pools of dATP, dTTP and incorporation of deoxythymidine into DNA during transformation. The increase in polymerase activity was paralleled by a similar increase in the pools of dATP and dTTP. The enzyme activity and the pool sizes of both nucleotides reached a maximum simultaneously with the peak of deoxythymidine incorporation into DNA. Studies in which protein synthesis was limited by cycloheximide showed that both the DNA polymerase activity and the rise in the pool sizes of both nucleotides were abolished. This implies that the de novo synthesis is required for the enzymes involved.

Human peripheral lymphocytes incubated in vitro have normally only little capacity for synthesizing macromolecules. However, addition of phytohemagglutinin (PHA) [l, 21 results in biochemical as well as cellular events so that the normal non-dividing lymphocyte is transformed into a blast-like dividing and metabolically active cell. Some of the early biochemical events in PHAstimulated lymphocytes are: an increased rate of 32P incorporation into membrane phospholipids a few minutes after addition of PHA to the cells [3]; an increase in acridine orange (AO) binding to deoxyribonucleoprotein [4]; stimulation of glycolysis [5, 61; increase in RNA synthesis [7]; acetylation of histones [8]. Later events are stimulation of protein [9] and DNA synthesis [lo]. For the last couple of years it has been demonstrated that PHA-stimulated lymphocytes show increasing DNA polymerase activity during

transformation, whereas the non-stimulated cells have only very little enzyme activity [l l-141. We found that this cell system might be suitable for evaluation of some control mechanisms involved in DNA replication. This communication deals with the determination of DNA polymerase activity in crude extract and the characterization of requirements and optimal conditions for the enzyme activity. Furthermore, a comparison has been carried out between crude enzyme preparations obtained from PHA-stimulated and non-stimulated cells. The interrelationship between DNA polymerase activity, incorporation of labeled deoxythymidine into DNA, and the pool size of deoxyadenosine triphosphate (dATP) and deoxythymidine triphosphate (dTTP) has been studied during transformation in the absence and presence of cycloheximide. Preliminary results have been presented elsewhere [ 151. Exptl

Cell

Res 77 ( 1973)

416

Gerda Tyrsted et al. MATERIALS

AND

METHODS

Chemicals Chemicals were obtained from following sources; unlabeled deoxyribonucleotides, ribonuclease A and cycloheximide; Sigma Chemical Co., St. Louis, MO; JH-deoxythymidine triphosphate, *H-deoxyadenosine triphosphate, 8H-methyldeoxythymidine (3H-TdR), 14C-L-leucine: New England Nuclear Corp.; aH-TdR (spec. act. 2 Ci/mmole) from The Radiochemical Centre, Amersham, UK; pancreatic deoxyribonuclease: Worthington Biochemical Corp.; actinomycin D: Calbiochem, Luzern; PHA-P from Difco Laboratories, Detroit, Mich.; tissue culture medium 199, penicillin, streptomycin and Hanks basal salt solution (HBSS); Flow Laboratories, Irvine, Scotland; Ficoll, Pharmacia, Uppsala, and Isopaque from NygPrd & Co., Oslo. Calf thymus DNA was prepared by the method of Hammarsten [16]; polymerase fragment from DNA nolvmerase from E. coli 1171 was kindly supplied byH.*Klenow of this institute. -

Isolation and incubation of human lymphocytes Peripheral blood was obtained from medical students and the lymphocytes were isolated by Beyums Ficoll-Isopaque technique [18] slightly modified [19]. The cells were cultured at a cell density of 1 x lo8 cells/ml in donors own serum and medium 199 supplemented with glutamine, penicillin and streptomycin [19]. Incubation was performed at 37°C in Fernbach flasks (Belco Glass Inc., N.J.) containing 65 or 20 ml cell suspension in 125 or 50 ml flasks, respectively. The contents of a commercial vial of PHA-P were dissolved in 50 ml HBSS and stored at - 18°C. The stimulatory effect of PHA, measured by 3H-TdR incorporation into DNA was confirmed for each new batch of PHA using varying concentrations. In the following experiments two batches of PHA were used and 20 ~1 PHA-solution per ml of cell suspension gave for both batches of PHA maximal stimulation between the second and third day after addition of PHA.

Determination of incorporation of precursors into DNA and protein The measurement of rate of incorooration of labeled deoxythymidine into DNA was performed by addition of 3H-TdR (suet. act. 12-15 Ci/mmole and 2 Ci/mmole, 0.5 &i/ml of cell suspension) to triplicate 2 ml lvmnhocvte cultures. The radioactivity incorporated into acid-insoluble product was determined after 1 or 2 h at 37°C by the filter paper technique 1201 with a minor modification [19]. The results are pre: sented as the means of triplicate determinations and exoressed as corn incoroorated ner lo6 cells nresent at-the beginning of the culture. The incorporation of deoxvthvmidine into DNA followed straight lines for at least i40 min. Exptl CeN Res 77 (1973)

The measurement of rate of incorporation of labeled amino acid into protein was done by addition of 14C-L-leucine (spec. act. 225 mCi/mmole, 0.5 &i/ml cell suspension) to an aliquot from a standard lymphocyte culture. The cells were transferred in 2 ml portions to 10 ml medicine bottles and the gas phase was equilibrated with atmospheric air containing 5 % CO,. Samples of 1.0 and 0.5 ml were removed at 0, 10, 20, 40 and 60 min after addition of isotope. The samples were added immediately to 2 ml ice-cold low2 M unlabeled leucine solution. After sedimentation for 15 min (500 g) at 4°C the supernatant was discarded and the tubes drained before adding 5 % trichloroacetic acid (TCA). The samples were heated to 90°C for 20 min and the precipitate was collected on Whatman GF/C glass fibre disks (25 mm in diameter). The tubes’ were rinsed with 3 x.2 ml TCA and filtered. The disks were washed with ethanol and ether and dried. The radioactivity was determined by liquid scintillation spectrometry. The rate of incorporated radioactivity followed straight lines for at least 2 h and was proportional to used amounts of cells. The results are expressed as cpm/ml of cells/h.

Preparation of enzyme extract and measurementof DNA polymerase activity After culturing for the desired length of time the cells were collected in 50 ml centrifuge tubes, sedimented for 15 min (500 g) at 4°C and washed once with cold HBSS. The tubes were drained and the cell pellet was resuspended at a cell concentration of 0.5-1.0 x 10’ cells in a buffer containing: 0.02 M potassium phosphate (pH 7.4), 20% w/c glycerol, a.1 mM dithiothreitol and 1 mM potassium EDTA. The cells were disrupted at 0°C with 1 min interval by sonication for 3 x 30 set with a MSE Mollard Ultrasonic disintegrator. The sonicate was centrifuged at 30000 g for 1 h. The low molecular comoounds were removed from the supematant by gel filtration chromatography performed with Sephadex G-25 medium. The gel column (1.5 x 21.5 cm) was pm-equilibrated with buffer (0.2 M Tris-HCl. oH 7.3. 20% w/v alvcerol. 0.1 mM dithiothreitol,’ 1 mM ‘potassium EDTA): The supernatant was applied in a volume of 3.6 ml. The column was eluted with the same buffer at 4°C and fractions of 0.94 ml were collected. The Sephadex chromatography gave rise to two peaks determined by the absorbance at 260 nm. For determination of total polymerase activity the fractions from the polymerase peak were pooled in a total volume of 8.4 ml. The measurement of enzyme activity was carried out the same day as the orenaration of the extract. The DNA polymerase assay measures incorporation of radioactivity from 3H-dTTP into an acid-insoluble product. The standard reaction mixture (330 ~1) contained: 12.0 pmoles Tris-HCl pH 7.3 at 22°C; 1.0 pmole KCl; 2.25 pmoles MgCl*; 0.3 pmole /?-mercaptoethanol; 12.5 nmoles each of dATP, dCTP, dGTP and dTTP; 5 pCi of 3H-dTTP (spec. act. 10-12 Ci/mmole); 150 pg calf thymus DNA; 100 ~1 eluate from Sephadex G-25 or crude sonicate. ‘Activated calf thymus DNA was prepared according to Apo-

Changes in some parameters shian et al. [21]. The incubation was performed at 37°C and the assay carried out with the filter paper technique [22]. Samples of 60 ~1 mixture were applied on Whatman no. 3 MM filter paper disks, 24 mm in diameter. The disks were immediately immersed in ice-cold 0.5 M nerchloric acid (PCA) (20 ml/disk) supplemented wiih 1.8 mM sodium tripolyphosphate. After rotating for 20 min the PCA was decanted and the washing was repeated twice for 10 min with PCA and 1.8 mM sodium tripolyphosphate, once with the same volume of ethanol sodium acetate (0.4 M sodium acetate in 93 % ethanol) and once with ethanol. The disks were dried and counted in a liquid scintillation spectrometer. All determinations were performed in duplicate or triplicate and the reaction rates are based on time samples taken at 0, 10, 20, 40 and 60 min. The enzyme activity is obtained as the differences between radioactivity incorporated with all four deoxyribonucleoside triphosphates present in the assay and the incorporation with a single deoxynucleoside triphosphate present. Unless otherwise indicated the activity is expressed as pmoles of radioactivity incorporated into acid-insoluble material per 30 min/lO’ cells incubated at the beginning of the cultures.

Extraction and determination of labeled DNA Isolation of labeled cellular DNA [23] was carried out after addition of 8H-deoxythymidine to an aliquot removed from a standard lymphocyte culture. The cells were transferred in 4 ml portions to 10 ml medicine bottles and the gas phase was equilibrated with atmospheric air containing 5 % CO*. A sample of 3.5 ml was removed at 0, 10, 20, 40 and 60 min after addition of isotope. On removal the sample was added to 1 ml ice-cold unlabeled 10L2 M deoxythymidine solution and centrifuged for 15 min (500 g) at 4°C. The cell pellet was extracted with 0.2 M PCA, sodium acetate in ethanol and ethanol-ether and digested for 18 h with 0.3 N KOH at 37°C. After precipitating DNA and protein with PCA, the supernatant was removed and the precipitate washed twice with 0.2 M PCA. DNA was extracted twice with 100 ~1 1 .O M PCA at 80°C for 20 min, and water was added to a final volume of 1 ml. All operations were performed at 0°C unless otherwise indicated. The amount of DNA was determined by the colorimetric method of Burton [24] with 2-deoxyadenylic acid as standard. This method measures only deoxyribose bound to purines and the results are not corrected for the pyrimidine content. The radioactivity was measured in a liquid scintillation counter. The incorporation of SH-TdR into DNA is defined as cpm/nmoles deoxyribose per hour obtained from the five time points which followed a straight line.

Other methods The pool size of dATP and dT’TP was determined enzymatically using a polymerase fragment from E. coli DNA polymerase and the copolymer of deoxyadenosine monophosphate and deoxythymidine monophosphate as a primer-template [19, 251.

involved in DNA synthesis

417

Protein was determined by the method of Lowry et al. [26] and bovine serum albumin was used ac standard.

RESULTS Requirementsfor DNA polymerase activity

DNA polymerase activity in crude or chromatographed extracts from non-stimulated or from PHA-stimulated cells which have not yet started transformation, as measured by incorporation of 3H-TdR into DNA, showed some activity in the absence of one or more of the other three deoxyribonucleoside triphosphates. With calf thymus DNA as template and in the absence of dATP. dCTP and dGTP the incorporation of 3HdTTP into acid-insoluble product was 2530% of that obtained for the complete assay with all four deoxyribonucleotides present (table 1). Omission of dGTP or of dGTP and dATP reduced the enzyme activity to about Table 1. Requirementsfor DNA polymeruse activity in crude extract from non-stimulated and stimulated l.ymphocytes pmoles dTMP incorporated in 30 min/lOB cells Cultures incubated without PHA

Cultures incubated with PHA

Reaction mixture

20h

44h

43 h

60 h -

Complete Minus dGTP Minus dGTP, dATP Minus dGTP, dATP, dCTP Minus DNA or Mg Complete + actinomycin D, 10 pg Complete + actinomycin D, 50 pg Minus 3 nucleotides -1~actinomycin D, 10 Pi3 Minus 3 nucleotides + actinomycin D,

5.2 2.3 1.5

2.8 1.8 0.7

36.5

59.8

1.3 0

0.6 0

0

8.9 0

50 c16

2.1 1.2 0.4

55.5 16.7 8.9

0.4 Exptl

Cell Res 77 (1973)

418

Gerda Tyrsted et al.

I

15.0 120 9.0 60 3.0

b k 20

60

100

V.0

160

Fig. 1. Abscissa: (a) ,uI enzyme extract; (b) ,ug added DNA: ordinate: DNA nolymerase activity. pmoles dTMP incorporated into-DNA in 30 min. - Effect of enzyme concentration (a) and various amounts of added primer (b) on DNA polymerase activity in chromatographed extracts prepared from lymphocytes incubated with PHA for 66 h. The enzyme assay was performed with 2 x lo8 cells/ml of extract.

50 and 30% of the control activity, respectively. With crude extract from stimulated cultures, incorporation of 3H-dTTP in the absence of the other three deoxyribonucleotides was about 15 % of the control activity (table 1). The same table shows that the presence of Mg2+ and DNA are necessary for the enzyme activity. In the presence of actinomycin D (10 or 50 ,ug) in the complete reaction mixture (table l), the polymerase activity in PHA-stimulated cells was 93 and 46 % of the control activity, respectively. With the same concentration of actinomycin the enzyme activity in non-stimulated cells was 75 and 43 % of that obtained for control cells. In the absence of the three deoxyribonucleoside triphosphates, no or very little inhibition with actinomycin D was found. Addition of pyruvate kinase, ATP and phosphoenolpyruvate to the assay mixture had no effect on the enzyme activity. On the other hand omission of mercaptoethanol reduced the activity about 10%. Characteristics of the DNA polymerase activity

Sephadex G-25 treated supernatant and crude extract from both stimulated and nonstimulated cells showed a pH optimum in Expti Cell Res 77 (1973)

Tris-HCl buffer at pH 8.0 at 22°C. The enzyme activity increased by about 30 % from pH 6.6 to 8.0 and by 10% from pH 7.3 to 8.0. Extract of stimulated cells treated with Sephadex G-25 was found to have the following properties of DNA polymerase activity. The enzyme activity shows to have linear relationship to the amount of enzyme (fig. la) and the effect of increasing DNA concentration on the DNA polymerase activity is represented in fig. 1 b. By digesting DNA with pancreatic DNase the primer effectivity increased less than 10%. On the other hand heat-denatured DNA (heated to 100°C for 10 min and cooled quickly to O’C) reduced the enzyme activity by 50 or 60 % in comparison with native and ‘activated’ DNA, respectively. The rate of incorporation of radioactivity from 3H-dTTP into an acidinsoluble product was linear for at least 60 min of incubation (fig. 2a). Fig. 2b shows that the DNA polymerase activity is maximal at a Mg2+ concentration of 7-9 mM. Similar curves were obtained with crude extract and the same characteristics were found both for stimulated and non-stimulated cells. The acid-insoluble product was found to be sensitive to DNase treatment, but not to RNase treatment.

10

30

50

2

6

10

14

16

22

Fig. 2. Abscissa: (a) min incubation; (b) mM MgCl,; ordinate: polymerase activity, pmoles dTMP incorporated into DNA in 30 min. Time course of the oolvmerase reaction (a) and the relationship between {he concentration of MgCl, and the DNA polymerase activity (b). The enzyme assay was performed with 2 x lo8 cells/ml of chromatographed extract prepared from lymphocytes treated with PHA for 66 h.

Changes in some parameters

involved in DNA synthesis

4 I9

Table 2. Comparison of DNA polymerase activity in different enzyme preparations and incorporation of 3H-deoxythymidine into DNA measured 2 h later than harvesting the cultures for enzyme preparation

Incubation for cells ______ h Expt 1 18 29 42

conditions

DNA polymerase activity (pmoles dTMP incorporated per lo6 cells)

in 30 min

PHA

Crude extract

3oooo g supernatant

G-25 eluate

DNA synthesis (incorporation of deoxythymidine) (cpm/2 h per lo6 cells)

3.2 11.5 39.9

2.7 13.9 39.2

2.8 13.3 43.6

847 33 034 55 385

1.6 27.2 54.3 26.4

2.0 24.0 49.0 20.8

1.3 24.9 49.8 23.1

240 14 886 25 266 17 450

4.4 2.5

4.5 2.3

3.9 2.1

229 106

I

Expt 2 18 42 8;

1

Expt 3 1:

-

A comparison was made between three types of enzyme preparations, crude sonicate, 30 000 g supernatant and pooled fractions from the polymerase peak after chromatography on a Sephadex G-25 column. The enzyme activity found in the 30 000 g pellet was about 5 % of that in the supernatant. It appears from the results in table 2 (representative from 7 experiments), that the enzyme activity observed in crude sonicate was the same as or higher than the activity in extracts which have been chromatographed. The same pattern was found for both stimulated and non-stimulated cells. Consequently, crude extract was suitable for determination of enzyme activity. To exclude the possibility that the low enzyme activity found in extracts from non-stimulated lymphocytes is caused by an inhibitor of DNA polymerase, the extract was mixed with extract from a stimulated culture. The results show (table 3) that there is neither evidence for an inhibitor in non-stimulated cells, nor for an activator in stimulated cells.

The DNA polymerase activity was studied in the presence of the sulfhydryl-blocking agent p-chloromercuribenzoate (&MB). In these experiments sulfhydryl-containing components were excluded from the reaction mixture and from the solutions used for ultrasonic treatment. Fig. 3 shows that Table

3. Effect of mixing Sephadex G-25treated extract from PHA-stimulated and non-stimulated lymphocytes Type of cells extracted

Mixing pmoles dTMP (relative incorporated volumes) in 30 min

Stimulated Non-stimulated Stimulated

None None 1

Non-stimulated Stimulated

1 i 1

Non-stimulated Stimulated

2 i 1

Non-stimulated

4

Expected values

14.6 1.6 7.4

8.1

5.4

6.0

4.0

4.2

Exptl Cell Res 77 (1973)

420

Gerda Tyrsted et al.

Fig. 3. Abscissa: (a, b) M p-chloromercuribenzoate; ordinate: (a) polymerase activity of extract from stimulated cells; (b) polymerase activity of extract from non-stimulated cells (semilogarithmic scale). The DNA polymerase activity is expressed as pmoles dTMP incorporated into DNA in 30 min/lOB cells. Effect of p-chloromercuribenzoate on DNA polymerase activity.

&MB is able to abolish the enzyme activity from stimulated as well as from non-stimulated cells. However, 50% inhibition of enzyme activity from stimulated cells is obtained at a pCMB concentration of 2.0 x 1O-s M, whereas the same inhibition from nonstimulated cells is found at a concentration of 2.0 x 1O-5 M. The effect of KC1 on DNA polymerase activity was investigated. In these experiments crude extract was used. The cells were sonicated in Tris-HCl buffer (0.02 M) containing sodium EDTA (1 mM), dithiothreitol (0.1 mM) and glycerol (20 % w/v). Addition of KC1 to the standard reaction mixture did not significantly increase the polymerase activity either at a total buffer concentration at 18 mM (not shown), or at 42 mM (fig. 4), however concentrations of KC1 beyond 25 mM were inhibitory. The enzyme from the stimulated cells appeared to be more inhibExptl Cell Res 77 (1973)

ited by high salt concentration than the enzyme from non-stimulated cells. The crude extract preparations kept frozen at - 18°C in the sonicating buffer solution were found to be unstable. In a week about 30 % of the activity was lost, consequently the polymerase activity was determined the same day as the preparation took place. In contrast to crude sonicate the chromatographed extract stored at the same temperature fell by only about 5 % during 3 weeks. The rate of deterioration of the enzyme activity of the chromatographed extract was determined at 37°C. At time intervals the extracts were assayed for polymerase activity with the usual technique and an enzyme preparation kept on ice served as control. The results are plotted on a semilogarithmic scale (fig. 5) and it appears from the curve that the decay of enzyme activity follows a first order reaction with t/2 equal to 120 min

0

60

120

180

240

Fig. 4. Abscissa: mM KCI; ordinate: polymerase activity in percent of controls without KC1 (semilogarithmic scale). Effect of the concentration of KC1 on DNA polymerase activity in crude extract prepared from lymphocytes incubated with PHA for 0, 60 h, and crude extract prepared from lymphocytes incubated without PHA for l , 20 h.

Changes in some parameters

l>

2

3

4

hours of incubation; ordinate: polymerase activity pmoles dTMP incorporated into DNA in 30 min (semilogarithmic scale). Effect of preincubation at 37°C on the DNA polymerase activity. The crude extracts were preincubated for various periods of time and then used in the usual assay conditions. Fig.

5. Abscissa:

with extract from a stimulated culture. The same decay was found at a ten fold increased dithiothreitol concentration. The protein content of the preincubated extracts was identical with that of the controls. When the same experiment was carried out with non-

involved in DNA

421

synthesis

stimulated extract the decay of enzyme activity followed a first order reaction with t/2 equal to 180 min. An experiment was then performed to see if any of the components in the standard reaction mixture could protect the enzyme from degradation during a 2 h period of preincubation at 37°C. The enzyme activity was determined both in the presence of all four deoxyribonucleoside triphosphates and in the presence of a single deoxyribonucleoside triphosphate. The presence of all four deoxyribonucleoside triphosphates gave a more pronounced decrease in enzyme activity in PHA-stimulated cells than in non-stimulated cells (table 4). On the other hand, in the presence in the enzyme assay of a single deoxyribonucleoside triphosphate, the reverse was observed., thus a greater decrease in enzyme activity was found in non-stimulated cells than in stimulated cells. If, however, the extract from stimulated cells was preincubated with DNA and magnesium separately or together, it was found that these components protected the enzyme equally well. A decrease of 20 Y/oor less was observed whether the assay mixture

Table 4. Effect of different components on DNA polymerase activity in crude cell extract prepared from lymphocytes incubated with PHA for 60 h and from lymphocytes incubated without PHA for 20 h Assay conditions PHA-stimulated Incubation conditions of extracts for 2 h 4°C 31°C

37°C DNA i- Mgz+ 37”C, Mg2+ 37”C, DNA 37”C, DNA + dGTP J dCTP + dATP

--Non-stimulated

extract

Presence of dATP, dCIP, dGTP, dTTP 85.4 32.1 71.9 67.0 72.5 48.9

extract

Presence of dTTP

Presence of dATP, dCTP, dGTP, dTTP

-Presence of d-fTP ____

14.4 7.8 18.6 12.4 13.4 14.8

6.0 4.3 10.9 6.0 8.2 5.4

1.7 0.4 5.9 1.8 1.7 1.8 --

The cells were from two individuals. The enzyme activity is expressed as pmoles dTMP incorporated in 30 min/106 cells, and was measured with all four deoxyribonucleoside triphosphates and a single deoxynucleotide (dlTP) present in the reaction mixture. Exptl

Cell

Res 77 (1973)

422

Gerda Tyrsted et al.

contained 1 or 4 deoxyribonucleoside triphosphates. In a similar experiment performed with extract from non-stimulated cells, it was observed that preincubation of extract in presence of DNA and magnesium stimulated the enzyme activity. On the other hand in the presence of DNA, dGTP, dCTP and dATP during preincubation the enzyme in non-stimulated cells was unaffected, whereas a decrease in activity was found in the stimulated cells when assayed in the presence of all 4 triphosphates. DNA polymerase activity during transformation and the correlation to the pool size of dATP and dTTP

The DNA polymerase activity, determined immediately after isolation of the lymphocytes, was found in the range 4-6 pmoles dTMP incorporated per 30 min per lo6 cells. This is higher than the activity found after incubation of the cultures for 18-20 h at 37°C (table 2). In the presence of PHA, incorporation of 3H-TdR into DNA began to increase about 18 h after the addition of PHA. The rate of DNA synthesis reached a maximum usually 50-60 h after addition of PHA. An increase of 150-600 fold was observed, compared with non-stimulated cultures in which incorporation of labeled deoxythymidine into DNA was in the range 100-400 cpm/Z h per lo6 cells present at the beginning of the culture. The DNA polymerase activity from cells treated with PHA increased in parallel with the rate of DNA synthesis and a peak value was also obtained between 50-60 h (fig. 6). An increase of 2030 fold was usually found, compared with the average enzyme activity observed in cultures incubated in the absence of PHA for about 18 h. We found it of interest to investigate the connection between the pool size of dATP and dTTP, the DNA polymerase activity and the rate of DNA syntheExptl

Cell Res 77 (1973)

66 60 52 44 36 20 20 12 4

Fig. 6. ordinate:

Abscissa: (a, b) hours of incubation with PHA; (a) pmoles dTTP per 10” cells (0); pmoles dATP per 10’ cells (e); (b, left) incorporation of *H-deoxythymidine into DNA; cpm x 1O-a per lo6 cells/2 h (e); (b, right) polymerase activity, pmoles dTMP incorporated in 30 min per lo8 cells (0). The correlation between the pool size of dTTP, dATP, DNA polymerase activity measured in crude extract and the incorporation of *H-TdR into DNA. The lymphocytes, from the same individual, were incubated for varying periods of time with PHA.

sis during transformation. However, the limiting amount of lymphocytes (3-4 x lo8 cell) which is normally obtained from one donor portion of blood prevents measurement of all parameters at early time points after addition of PHA, since the non-stimulated lymphocyte has very samll pools of dATP and dTTP [19]. Therefore the determinations were carried out between 24-91 h after addition of PHA. The cells, isolated from one donor in a total number of 7.8 x 10s, were incubated with PHA. After the desired length of time of incubation, a number of cultures sufficient for the required determination were pooled. Labeled deoxythymidine was added to an aliquot of the

Changes in some parameters

Table 5. Effect of 0.2 pg/ml cycloheximide extract,

W-leucine

Treatment with

and 3H-deoxythymidine

Leucine incorporation

Cycloheximide cpm/ml from per h

involved in DNA synthesis

423

on DNA polymerase activity measured in crude incorporation into protein and DNA, respectice1.y

Deoxythymidine

incorporation

DNA-polymerase activity pmoles dTMP incorporated in 30 min

% of control

cpm/ml per h

% of control

w-4 DNA per h

y, of control

per DNA

9; of control

“” of per lo6 cells control -~

(100) 31

25 190 2160

(100) 9

518 63

‘y

3.9 0.7

(1;)

45.1 5.6

(100) 12.

O-694 h None 3 480 O-694 h O-69& h 2 940

Y?

18916 20 379

(100) 108

392 531

‘2

3.5 3.1

‘1~~

42.2 28.3

(100) 67

Expt 2 W5 h 0-45h

None 0-45h

“gl

11 250 150

(100)1

407 50

(1;)

;;

w$)

18.2 3.3

w$

O-45 &69 o-69 o-69

3045 h 725 None 2 370 O-69 h 1450 30-69 h 1305

T

5 050 l9 553 11441 7 468

45 (Y

41 ‘1;;)

1:X 3.7 2.7 2.5

69 (1;)

38

178 630 466 260

11.3 36.8 17.0 19.7

63 (100) 46 54

None O-93 h

“2

24 639 10199

(100) 41

814 431

(1:)

5.0 4.3

“gl

50.9 28.7

(100) 56

PHA from

Expt 1 o-45 h None 045 h O-45 h

h h h h

O-93 h O-93 h

3 640 1140

1 ;gy

2 795 1 405

40 55

The isotopes were added to the cells 1 h prior to terminating expressed as nmoles deoxyribose (see Methods).

cells 2 h prior to harvesting the cells for measurement of the pool size and polymerase activity. Fig. 6a illustrates the variation in the pools of dTTP and dATP and fig. 6b illustrates the DNA polymerase activity and incorporation of 3H-deoxythymidine into acid-insoluble material during transformation. From the experiment it appears that between 24-76 h the variation in the pools of both deoxyribonucleotides is paralleled by a similar variation in polymerase activity and rate of DNA synthesis. The peak values coincide at about 50 h after addition of PHA. However, in the time period between 76-91 h the pool size of dTTP and dATP is increasing, the rate of DNA synthesis is decreasing and the polymerase activity is constant. The increase in pool size of dATP and dTTP about 80-90 h after addition of PHA and the finding that the pool size of dTTP exceeded that of dATP during trans-

41

68

the cultures for enzyme determination,

DNA is

formation, has been observed in other experiments [ 191. Inhibition of protein synthesis by cycloheximide

By inhibition of protein synthesis, it should be possible to determine whether the induced rise in DNA polymerase activity and the enlargement of the pools of dATP and dTTP during transformation was due to synthesis of new enzyme proteins or activation of existing enzyme molecules. For this purpose cycloheximide was used at a concentration of 0.2 ,ug/ml of cell suspension, thus the protein synthesis was inhibited about. 70% measured by incorporation of 14C-leucine. Table 5 shows the effect of cycloheximide on DNA polymerase activity and on the incorporation of labeled precursors into protein and DNA. In the first experiment cycloheximide and PHA were added simulExptl Cell Res 77 (1973)

424

Gerda Tyrsted e tal.

Table

6. Incorporation of 14C-leucine into protein expressed on basis of cell number, 3H-deoxythymidine incorporation into DNA expressed on basis of cellular DNA content, and the pool size of dTTP and dATP in presence of 0.2 ,ug cycloheximide per ml of cell suspension Treatment with

Leucine SH-TdR pmoles dTTP Cycle- incorp. incorp. heximide % of % of per lo6 % of from control control cells control

PHA from Expt

pmoles dATP per DNA

% of control

per lo6 % of cells control

per DNA

% of control

2.9

0.33 0.06

(1;)

0.33 0.21

‘1;)

8::

(lo$l

I

0-47h G47h o-68 h o-68 h O-74 h o-74 h O-97 h O-97 h Expt

2

tS68 O-68 O-68 O-68

h h h h

z?eh None O-68 h

(loo) 34 (100) 97

None h o-74

T

z;“h

(??

65-68 h 62-68 h

43 47

0.91 0.17 0.86 0.49 0.69 0.50 0.19 0.28

52

6.1

113 87

0.72 0.51 0.83 0.69

0.4 2.9 1.7 4.8 2.5 1.6 1.2

‘1;)

5.6

(10$

115 96

i: 3:8

84 68

The isotooes were added 1 h orior terminating the cultures for determination as nmoles deoxyribose (see Methods). -

taneously to the cultures, and it appears that the presence of cycloheximide for 45 h inhibited the rate of protein synthesis, DNA synthesis and polymerase activity. After 69.5 h the cultures were able to transform, measused by increasing rate of protein synthesis, DNA synthesis and enhancement in polymerase activity, which in some experiments exceeded the values in the control cultures. A similar pattern was found from other experiments, as the presence of cycloheximide inhibited the protein and DNA synthesis and DNA polymerase activity for approx. 2 days, but between the 2nd and 3rd day these parameters were increasing. In the second experiment some of the cultures were stimulated for 30 h before cycloheximide was added. The presence of cycloheximide in the cultures for 15 and 39 h reduced the polymerase activity related to cellular DNA content by Exptl

Cell Res 77 (1973)

0.14 0.13 0.58 0.23 0.49 0.44

84 76

of the pools. DNA is expressed

approx. 30%. In this experiment an atypical transformation took place due to handling the control cultures in the same way as the cycloheximide-treated cultures. It is well known that the transformation pattern can change if the cultures are shaken. To avoid the error due to cell death or fluctuation of cell number during prolonged cycloheximide treatment, cellular DNA content was measured. The amount of DNA varied in the range 63-98 Y0 of that of the controls, dependent on the duration of cycloheximide treatment. This variation in cell number is reflected in the results in tables 5 and 6, in which polymerase activity and pool size of dATP and dTTP are expressed both per number of cells incubated at the beginning of the cultures and per DNA content. It appears that the results expressed per DNA content always either exceed or are the same as the

Changes in some parameters

corresponding results expressed on the basis of cell number. Table 6 shows the rates of protein and DNA synthesis and the pool size of dTTP and dATP in cycloheximide-treated cultures. In the first experiment cycloheximide was added to the cultures simultaneously with PHA. It appears from the results that the pool size of dTTP and dATP was approx. 20 % of that of the control cells in the first 2 days in the presence of cycloheximide, thereafter an enlargement in the pools was observed. In the 2nd experiment cycloheximide was present in the cultures for 68 h, 3 h and 6 h. The rates of protein and DNA synthesis were decreased about 50% for the three time points. During 3 h and 6 h treatment with cycloheximide the variation in the amounts of dTTP was small compared to the control. On the other hand, cycloheximide had a greater effect on the pool size of dATP which was decreased. In addition to the parameters shown in this experiment, the activity of DNA polymerase was measured after 68, 3 and 6 h treatment with cycloheximide. The enzyme activity amounted to 46, 86 and 87 “/b of the activity in a 68 h PHA-stimulated culture, expressed on the basis of cellular DNA. The corresponding percentages based on cell number were 30, 84 and 80 %. DISCUSSION The DNA polymerase activity in crude extract or Sephadex G-2%treated extract prepared from PHA-stimulated and non-stimulated human lymphocytes has been characterized, and we found the same requirements as Loeb et al. [ll]. The findings were the same for extract of PHA-stimulated cells and non-stimulated cells, and no differences were found between crude extract and chromatographed extracts. In addition, we have con-

involved in DNA

synthesis

425

cluded from the results in table 2 that it is not advantageous to use chromatographed extract instead of crude extract as enzyme source since the data imply that during transformation the nucleotides do not interfere with measurement of DNA polymerase activity. In the absence of dGTP, dCTP and dATP in the reaction mixture, we found a difference between polymerase activity in extract prepared from cells which had started transformation and in extract from nonstimulated cells (table 1). A higher enzyme activity was observed in the latter. This lack of complete dependence on the presence of all four deoxyribonucleoside triphosphates is in agreement with results which describe terminal deoxynucleotidyl transferase activities in nuclear and soluble extracts of calf thymus [27-281. In addition, the polymerase activity was inhibited in the presence of actinomycin D and the four deoxynucleoside triphosphates in the reaction mixture, whereas in the presence of a single deoxynucleoside triphosphate the drug had little or no effect on the enzyme activity (table 1). This observation supports the above interpretation, since actinomycin D inhibits the replicative DNA nucleotidyl transferase activity to a higher extent than the terminal enzyme activity [29]. The sulfhydryl blocking agent (pCMB) inhibited the polymerase activity. Thus, 50 % inhibition of enzyme activity in stimulated cells was found at a tenfold lower pCMB concentration than in non-stimulated cells. There have been several reports dealing with the influence of monovalent cations on the activity of DNA polymerase isolated from bacteria [30, 311 and from mammalian cells [29]. In the latter the enzyme activity is increased by low concentrations of K + and inhibited by higher concentrations. From fig. 4 it appears that Kf at concentrations up to about 25 mM had a very modest stimulatExptl

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Rar 77 ( 1973)

426

Gerda Tyrsted et al.

ing effect on the polymerase activity, and 50 % inhibition of enzyme activity from stimulated cells and from non-stimulated cells was obtained at 85 mM KC1 and 130 mM KC1 respectively. The activation of DNA polymerase activity by K+ is probably dependent on the type of buffer and ionic strength [30, 311. If extract from non-stimulated cells was preincubated with Mg2+ and DNA (table 4) an increase in enzyme activity was observed, both when all four or a single deoxyribonucleoside triphosphate was added to the reaction mixture. This observation cannot be interpreted at the moment, but may explain why in non-stimulated cells, we find that the enzyme activity in the presence of a single deoxyribonucleoside triphosphate constitutes a greater percentage of the enzyme activity when all four deoxyribonucleoside triphosphates are present, by comparison with the corresponding enzyme activities in stimulated cells. We observed a considerable DNA polymerase activity in normal lymphocytes without incubation, but during a day of incubation the enzyme activity decreased when expressed per number of cells originally placed in the cultures [14]. Addition of PHA gave usually 20-30 fold stimulation of polymerase activity compared with cultures incubated for 18-20 h, an increase which is of the same magnitude as found by others [14]. The increase in DNA polymerase activity during transformation was paralleled by an increase in the rate of incorporation of labeled deoxythymidine into DNA, and similarly an increase in the pool size of dATP and dTTP was found. The increase in polymerase activity and in the pools of dATP and dTTP could be abolished by cycloheximide, indicating a de novo synthesis of enzyme proteins during transformation. Determination of the pool size of all four Exptl Cd Res 77 (1973)

deoxynucleoside triphosphates during transformation under different conditions is necessary before evaluation can be made of the control mechanisms involved in the regulation of the pools. Ribonucleoside diphosphate reductase [32] might possibly be of importance in controlling these pools. On the other hand, Rabinowitz et al. have clearly demonstrated in normal and leukemic lymphocytes [33] and in lymphocytes cultured with PHA [34] that the formation of dTTP from thymidine depends on ATP concentration, thus changes in ATP concentration could exert a controlling influence on the TdR Salvage pathway and then on the pool size of dTTP. We are grateful to Professor Hans Klenow for his interest and for reading the manuscript. The blood was drawn from medical students through the courtesy of Dr Bo DuPont. The skilful technical assistance of Mrs Jannie Jensen, Mrs Inger Lyhne and Mrs Gunhild Olesen is gratefully acknowledged. We wish to thank Miss Birthe Rasmussen for her excellent drawings. This work was supported in part by Landsforeningen til kraeftens bekaempelse, Anders Hasselbalchs fond til leukemiens bek2empelse, Fonden til laegevidenskabens fremme, Kong Christian d. X’s fond, F. L. Smidth og Co., A/S Jubileumsfond, Statens hegevidenskabelige og naturvidenskabelige forskningsrad.

REFERENCES 1. Hungerford, D A, Donelly, A J, Nowell, P C & Beck, S, Am j hum genet 11 (1959) 215. 2. Nowell, P C, Cancer res 20 (1960) 462. 3. Fisher, D B & Mtiller, G C, Proc natl acad sci US 60 (1968) 1396. 4. Killander, D & Rigler, R, Exptl cell res 39 (1969) 710. 5. Cooper, E H, Barkhan, P & Hale, A J, Brit j haematol 9 (1963) 101. 6. Rabinowitz, Y, Schimo, J & Wilhite, B A, Brit j haematol 15 (1968) 455. 7. Cooper, H L, Proc 5th leucocyte culture conf (ed J E Harris) P. 15. Academic Press, New York (1970). 8. Pogo, B G T, Allfrey, V G & Mirsky, A E, Proc natl acad sci US 55 (1966) 805. 9. Ling, N R, Lymphocyte’ stimulation (ed N R Ling) p. 201. North-Holland, Amsterdam (1968). 10. - Ibid. p. 175. 11. Loeb, L A, Agarwal, S S & Woodside, A M, Proc natl acad sci US 61 (1968) 827.

Changes in some parameters 12. Loeb, L A, Ewald, J L & Agarwal, S S, Cancer res 30 (1970) 2514. 13. Loeb, L A & Agarwal, S S, Exptl cell res 66 (1971) 299. 14. Rabinowitz, Y, McCluskey, I S, Wong, P & Wilhite, B A, Exptl cell res 57 (1969) 257. 15. Tyrsted, G, Munch-Petersen, B & Cloos, L, Exptl cell res 67 (1971) 259. 16. Hammarsten, E, Biochem Z 144 (1924) 383. 17. Klenow, H & Overglrd-Hansen, K, FEBS letters 6 (1970) 25. 18. Boyum, A, Stand j clin lab invest, suppl. 21 (1968) 77. 19. Munch-Petersen, B, Tyrsted, G & DuPont, B, Exptl cell res. In press. (1973). 20. Sorensen, S F, Andersen, V & Giese, J, Acta path microbial Stand 76 (1969) 259. 21. Aposhian, H V & Kornberg, A, J biol them 237 (1962) 519. 22. Bollum, F J, J biol them 234 (1959) 2733. 23. Munro, H N & Fleck, A, Methods of biochemical analysis (ed D Glick) vol. 14, p. 113. Wiley, New York (1966). 24. Burton, K J, Biochemistry 62 (1956) 315.

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25. Lindberg, U & Skoog, L, Anal biochem 34 (1970) 152. 26. Lowry, 0 H, Rosebrough, N J, Farr, A I, & Randall, R J, J biol them 193 (1951) 265. 27. Keir, H M & Smith, M J, Biochim biophys acta (1963) 589. 28. Bollum, F J, Groeniger, E & Yoneda, M. Proc natl acad sci US 51 (1964) 853. 29. Keir, H M, Progr nucleic acid res molec hiol 4 (1965) 81. 30. Klenow, H & Henningsen, 1, Eur j hiochem 9 (1969) 133. 31. Bishop, C C, Jr & Gill, J E, Biochim biophys acta 227 (1971) 97. 32. Larsson, A & Reichard, P, Progr nucleic acid res molecular biol 7 (1967) 303. 33. 7Rgbinowitz, Y & Wilhite, B A, Blood 33 t 1969) 34. Rabinowitz, Y, Wong, P & Wilhite, B A, Blood 35 (1970) 236. Received July 11, 1972 Revised version received August 14, 1972

Exptl

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