Proc. Nat. Acad. Sci. USA Vol. 72, No. 6, pp. 2413-2417, June 1975

Viral and Cellular DNA Synthesis in Nuclei from Human Lymphocytes Transformcd by Epstein-Barr Virus (DNA virus/tumor virus/isolation of nuclei)

WENDY C. BENZ AND JACK L. STROMINGER The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138; and The Sidney Farber Cancer Center, Harvard University, Boston, Massachusetts 02115

Contributed by Jack L. Strominger, March 10,1975 A DNA-synthesizing system in vitro, ABSTRACT using nuclei prepared by treatment of human lymphocytes with the detergent Brij 58, was developed. Nuclei from cultured lymphocytes synthesized DNA for as long as 5 hr, and required ATP, deoxynucleoside triphosphates, magnesium, and a calcium chelator. In nuclei from a partially synchronized line of cultured lymphocytes carrying several hundred copies of the Epstein-Barr viral genome, synthesis in vitro was predominantly viral in early S phase and cellular in late S phase. These and other data suggested that the DNA synthesis observed in vitro was predominantly replicative.

)NA synthesis has been studied in a variety of eukaryotic cell types in culture, either in intact cells or in isolated nuclei, with both normal cell lines and cell lines infected by several DNA viruses, notably tumor viruses (for example, refs. 1-11). The study of both tumor virus and host DNA synthesis in virally transformed cells (as contrasted to productively infected cells) is made difficult by the problem of distinguishing the two processes. One cell system that would be suitable for such studies is human lymphocytes transformed by Epstein-Barr virus (EBV). There are available a wide range of lymphocyte lines that carry varying numbers of EBV genomes (12, 13). Furthermore, some of these are producer lines, i.e., a small portion of the population of cells is undergoing viral macromolecular synthesis at all times; others are nonproducer lines, in which viral DNA and antigen synthesis can be induced only by chemicals or superinfection (14-16). DNA synthesis in transformed lymphocytes is of particular interest for an additional reason. Normal peripheral lymphocytes are resting cells in terms of DNA synthesis, and EBV has been shown to cause them to undergo transformation into cells which now synthesize DNA and divide for an indefinite number of generations (17-19). It is, therefore, possible that the presence of the viral genome or some viral gene product (such as the Epstein-Barr nuclear antigen, EBNA) affects the control of host DNA synthesis in order for the cel] to assume the transformed state. Study of DNA synthesis in isolated nuclei has many advantages over its study in intact cells. However, lymphocyte nuclei are difficult to prepare in an active state after mechanical disruption in the Dounce homogenizer, the usual method for preparing nuclei, probably because their relatively large size renders them sensitive to damage. In the present paper a modified procedure for making lymphocyte nuclei in the presence of detergent without mechanical disruption of the cells (20, 21) is reported. These nuclei are active in a DNAsynthesizing system in vitro over a long period of time. Some characteristics of host and viral DNA synthesis in these lreparations will be reported. Abbreviation: EBV, Epstein-Barr virus. 2413

MATERIALS AND METHODS

Lymphocyte Nuclei. Cell lines Raji, IM-1, RPMI 4265, P3HR-1, and Molt (an EBV-negative T cell line) were obtained from Drs. A. Adams, G. Klein, H. zurHausen, D. Mann, and J. Minowada. Cultures were grown in RPMI 1640 medium containing 10% fetal calf serum, penicillin, and streptomycin (ABS) in closed static suspension culture at 37°. Lymphocytes from freshly drawn human blood were prepared by the Ficoll-hypaque density gradient centrifugation method (22) and washed in dextrose/gelatin/ veronal Buffer (GIBCO). Cells were also washed in RPMI 1640 medium three times and placed in culture with 10 ,g/ml of phytohemagglutinin (Difco) in RPMI 1640 medium containing 10% fetal calf serum, penicillin, and streptomycin at 2 X 106 cells per ml for 40-65 hr at 37°. The method of preparing lymphocyte nuclei in the presence of detergent (20, 21) was modified as follows. Cultured lymphocytes in logarithmic phase, phytohemagglutininstimulated lymphocytes, and fresh peripheral lymphocytes prepared as described above were sedimented out of the culture medium or buffer, and washed once at room temperature with a buffer of 0.15 M sucrose, 5 mM CaCl2, 25 mM Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 8.0. The cells were then gently resuspended in 0.5-1.0 ml of a buffer of 0.25 M sucrose, 5 mM CaCl2, 25 mM Hepes, pH 8.0, chilled, and diluted with an equal volume of a chilled solution of 0.15 M sucrose, 5 mM CaCl2, 25 mM Hepes, pH 8.0, and 0.5% Brij 58 (Atlas) or other detergents. The cells were then gently agitated on ice for 5 min. They were diluted 20- to 30-fold with the chilled 0.25 M sucrose buffer described above containing 2% Dextran type C 100 (Sigma), pelleted in the cold, and resuspended to a final concentration of 2 X 101 to 2 X 107 per ml with the same chilled buffer. The resultant nuclei were counted in a hemacytometer and used immediately. DNA Synthesis In Vitro. The following incorporation mixture (3, 4) was made freshly for each experiment: 0.5 mM in each of dATP, dGTP, and dCTP (Sigma); 0.2 mM [3H]dTTP (50-100 /ACi/umol, Schwarz/Mann); 5.0 mM ATP (Sigma), 12.5 mM phosphoenolpyruvate, 5 U/ml of pyruvate kinase (Sigma), 38 mM Hepes buffer pH 8.0, 0.125 M sucrose, 0.1 M NaCI, 12.5 mI 1\IgCl2, 12.5 mM ethyleneglycol bis(0aminoethyl ether)N,N'-tetraacetic acid (EGTA), 2 mM dithiothreitol. A 0.1-ml aliquot of this mixture was mixed with 0.15 ml of nuclei (3 X 105 to 3 X 101 nuclei) and incubated at 37°. Incorporation of acid-insoluble radioactivity was determined by trichloroacetic acid precipitation, with GF/A or GF/C filters (Whatman) (23); a liquid scintillation spectrometer was used.

Microbiology: Benz and Strominger

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FU 600 )- TRANSFORMED LYMPHOCYTES (RA)

0

D (240O z 0

x

UO

' 4=C

2 ( 160) E E 200 a (80)

PHA-TREATED LYMPHOCYTES

FRESH PERIPHERAL LYMPHOCYTES

2 2 3 HOURS OF INCUBATION (370)

4

5

FIG. 1. Time course of incorporation of radioactivity from [3H]dTTP by nuclei. Nuclei from Raji cells in early logarithmic phase, and from phytohemagglutinin (PHA)-stimulated and normal fresh peripheral lymphocytes, were each incubated with the DNA synthesis mixture described in Materials and Methods.

Alkaline Sucrose Gradients. Nuclei were incubated at 370 in the incorporation mixture described above, except that the [3H]dTTP was now 5 ,uM (10 mCi//umol, New England Nuclear). Samples were removed and diluted into several volumes of chilled buffer (0.25 M sucrose, 5 mM CaCl2, 25 mM Hepes, pH 8.0, 2% Dextran C 100); they were pelleted and washed twice more in the same chilled buffer. They were lysed in 0.2 M NaOH, 10 mM EDTA at 370 for 4-6 hr, layered onto 12-ml gradients of 5-20% sucrose, 0.9 M NaCl, 0.1 M NaOH with a saturated CsCl cushion, and sedimented in an IEC SB283 rotor at 60,000 X g for 15 hr at 20C. About 30 TABLE 1. Effect of different detergents on recovery of nuclei and DNA-synthesizing activity

Detergent* 0.25% Nonidet P-40/0.125% desoxycholate 0.25% Triton X-100/0.125% desoxycholate 0.25% Atlas G 2090 0.25% Brij 96 0.25% Brij 58 0.25% Tween 21 0.25% Span 20 0.25% Span 40 Dounce homogenization after hypotonic swelling

DNA Recovery synthesis of (cpm/5 X 106 HLB nuclei (%) nuclei) 13.5

35

1900

13.5 12.5 12.4 15.7 13.3 8.6 6.7

40 30 35 60 35 40 40

1300 1900 3300 2500 150 60

20

170

Nuclei from Raji or IM-1 cells were prepared as indicated in Materials and Methods, except that the detergents listed above substituted for Brij 58 at the indicated concentrations. Cells and nuclei were counted with a hemacytometer. Some preparations were placed in the DNA synthesis mixture for 60 min at 37°. HLB, hydrophobic-lipophilic balance number. * In addition, nuclei prepared with the following detergents were not tested for DNA synthesis: Nonidet P-40 (HLB 13.5, 3% yield of nuclei), Triton X-100 (13.5, 10%), Triton X-114 (12.4, 3%), Brij 76 (12.4, 30%), and Tween 81 (10.0, 30%).

were

(1975)

fractions per 12-ml gradient were collected. To each fraction, 0.5 ml of calf thymus DNA (0.1 mg/ml) in 50 mM Tris HCl (pH 8.5), 5 mM EDTA was added, and each tube was then shaken on a Vortex with 3.0 ml of chilled 5% trichloroacetic acid with 33 mM sodium pyrophosphate. After 10-15 min in the cold, the fractions were then poured onto GF/C filters, washed with trichloroacetic acid/pyrophosphate, then with 10 mM HCI, dried, and counted in a scintillation counter. 82P-Labeled DNA fragments of lambda ph3ge of measured length were a gift of T. Maniatis. Synchronization of Transformed Lymphocytes. The double thymidine block procedure for cell synchronization was done as described (24), except that 8 mM thymidine was used for P3HR-1 cells, and 2 mM thymidine was used for Molt cells. Neutral CsCl Gradients. Preparation of nuclei for the gradients and the conditions of the gradients were as described (25). Sedimentation was performed in an IEC rotor SB110 at 50,000 X g for 70 hr at 200. Marker EBV [IH]DNA was a gift of Dr. C. Crumpacker. In experiments where marker [3H]DNA was used, nuclei were labeled with [a-32P]dTTP (New England Nuclear, used at 1 mCi//umol). About 40 fractions per gradient were collected, and each fraction was treated with trichloroacetic acid/pyrophosphate as described above. RESULTS

Preparation of Lymphocyte Nuclei by Detergent Treatment. Although procedures for the preparation of lymphocyte nuclei using the 1)ounce homogenizer have been reported (26), the large size of the nucleus may render these cells more susceptible to mechanical disruption than other cell types. To avoid problems of disruption, a previously reported method of making nuclei based on detergent treatment in the presence of a high sucrose concentration (20, 21) was modified and tested with many detergents covering a wide range of hydrophobic-lipophilic balance (HLB) numbers. The most hydrophilic detergent tested, Brij 58, gave the highest recovery of nuclei from whole cells (Table 1) and also, as described below, the highest level of DNA-synthesizing activity. It was used routinely in subsequent experiments unless otherwise stated. Measurement of DNA Synthesis in Detergent-Treated Nuclei. A mixture of four deoxynucleoside triphosphates, ATP, magnesium, and other components described in Mlaterials and Methods was incubated with the nuclei from transformed lymphocytes prepared by Brij 58 treatment. DNA synthesis was measured as incorporation of radioactivity from [3H]dTTP into acid-insoluble material. Unlike reported systems for other cell types using mechanical disruption, nuclei from detergent-treated transformed cells in early logarithmic phase utilized [3H]dTTP for up to 5 hr (Fig. 1); even nuclei prepared from cells in midlogarithmic phase continued incorporation for at least 3.5 hr. These nuclei did not incorporate radioactive label from added [3H ]thymidine. A rough comparison of the rate of DNA synthesis in transformed cells in vivo and in vitro was based on the following calculations. With approximately 6 pg of DNA per cell (27) and a generation time of 24 hr under existing culture conditions, DNA synthesis in Raji cells in vivo proceeded at a rate of about 1.5 X 10-3 pmol of DNA per 20 min per cell. Assuming that approximately 25% of isolated nuclei obtained from logarithmic phase cells were in S phase and that the rate of DNA synthesis was four times the rate of dTMIP incorpora-

Proc. Nat. Acad. Sci. USA 72

DNA Synthesis in Lymphocyte Nuclei

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tion, then from the data in Fig. 1 it was calculated that at early times DNA synthesis proceeded at a rate of about 2.4 X 10-4 pmol/20 min per nucleus, or about 1/6 of the rate in vivo. This estimate of the rate in vitro was a minimal one, since it did not take into account dilution of isotope by possible intranuclear pools of unlabeled dTTP. Deoxyribonucleoside triphosphates, ATP, magnesium, and EGTA were strong requirements for DNA synthesis (Table 2). The ribonucleoside triphosphates did not substitute for the deoxyribonucleoside triphosphates, nor did various other ribonucleoside triphosphates completely fulfill the ATP requirement (Table 3). Inclusion of GTP, UTP, and CTP in addition to ATP and the deoxynucleoside triphosphates gave a small enhancement of incorporation (10-20%), particularly evident at later times and reproducible in four experiments using different nuclear preparations. Nuclei prepared in the presence of Brij 58 gave better DNA-synthesizing activity than those prepared with the other detergents (Table 1). Nuclei prepared after treatment with hydrophobic detergents were inactive for DNA synthesis, although good yields of nuclei were obtained. Mechanical disruption of cells by the Dounce homogenizer also gave inactive nuclei. All of the results reported in Tables 1-3 are for nuclei from the Raji and IM-1 lines of human lymphocytes. However, DNA synthesis with nuclei from detergent-treated cells was also successfully carried out with the RPMI 4265, Molt, and P3HR-1 cell lines. DNA Synthesis in Nuclei Prepared from Detergent Treatment of Peripheral and Phytohemagglutinin-Stimulated Lymphocytes. In nuclei from phytohemagglutinin-treated cells, DNA synthesis continued for less than 60 min and proceeded at a slower rate than in nuclei from transformed cells. Nuclei from fresh lymphocytes not treated with phytohemagglutinin showed no significant DNA synthesis in this system (Fig. 1). These latter data are in contrast to findings reported by Fridlender et al. (26) and will be discussed below. Also, in contrast to their report, the incorporation of radioactivity from [3H]dTTP in nuclei from phytohemagglutinin-stimulated lymphocytes in the present study showed a strong requirement for ATP, since absence of ATP and the generating system gave only 23% of the incorporation in the control (complete system, using a 60-min incubation, 1150 cpm; ATP omitted, 264 cpm per 5 X 106 nuclei). Utilization of BrdUTP for DNA Synthesis in Isolated Nuclei. To determine whether short repair segments or longer replicative segments (28) were being synthesized tn vitro, logarithmic phase Raji cells were allowed to incorporate [3H]thymidine for several minutes in vivo. Nuclei were then prepared by Brij 58 treatment, allowed to synthesize DNA in an incorporation mixture in which [3H]dTTP had been replaced by bromodeoxyuridine triphosphate (BrdUTP, P-L Biochemicals), and lysed. The DNA was sheared by six passages in a 21-gauge needle and sedimented in neutral CsCl density gradients. After 50 min of synthesis in vitro, approximately one quarter of the total DNA present was significantly denser than the DNA synthesized in vivo (Fig. 2), while only a small amount of this material was seen at 15 min. Presumably the separation of 3H label incorporated in vivo and heavy label incorporated in vitro was due to the low counts of 3H incorporated (making it difficult to detect a small amount of this associated with the heavy label) and of shearing of the DNA to relatively small size. Sedimentation of sheared material in

2415

TABLE 2. Requirement for components for DNA synthesis by nuclei DNA synthesis (cpm/5 X 106 nuclei) 60 min 180 min

System

Complete system -Dithiothreitol

-NaCi -EGTA

-MgC12 -ATP and ATP generating system -ATP generating system -dATP -dGTP -dCTP

2680 2780 2600 330 220 480 1760 830 520 530

5740 4200 6870 860 230 550 2780 860 1490 920

Raji or IM-1 nuclei were incubated at 370 in the DNA synthesis mixture. Components were omitted as indicated.

alkaline sucrose gradients indicated that its average size was between 5 X 106 and 1 X 107 daltons. These observations were consistent with the interpretation that longer segments of DNA rather than short segments were being made, suggesting that the process was replicative rather than repair synthesis. However, a long patch repair process (28) could not be entirely ruled out by this experiment. Incorporation of Radioactive Label into Small Material on Alkaline Sucrose Gradients. Experiments were performed with nuclei from Brij-58-treated, transformed lymphocytes to look for the presence of small, single-stranded DNA fragments on alkaline sucrose gradients. These have been reported in prokaryotic (29) and eukaryotic (e.g., refs. 2, 7, and 9) cells undergoing DNA synthesis, and are considered to be an intermediate in the discontinuous mode of DNA replication. Such small material appeared after a pulse of ['H]dTTP in vitro, and was chased after addition of an excess of unlabeled dTTP (see Fig. 3B in ref. 30). Further experiments with alkaline gradients in which DNA was sedimented further in the presence of two DNA markers of known base length showed peaks of small material sedimenting approximately TABLE 3. Effect of substitution or addition of ribonucleoside triphosphates on DNA synthesis DNA synthesis

(cpm/5 X 106 nuclei) Substitutions

60 min

180 min

Complete system ATP for dATP GTP for dGTP CTP for dCTP ATP, CTP for dATP, dCTP GTP for ATP CTP for ATP UTP for ATP dATP for ATP Addition of GTP, CTP, and UTP at 0.3 mM each

3260 1460 1370 390 490 1350 360

6170 1730

2450

1050

1120

3380

7400

2530 2160 870

550 1460

Raji nuclei were incubated at 37' in a DNA synthesis mixture

in which the components

were

varied

as

indicated.

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Microbiology: Benz and Strominger

C.4.5HR

BOrTTO

FRACTIONS

~I

Proc. Nat. Acad. Sci. USA 72

TOP

FIG. 2. Neutral CsC1 gradients of sheared DNA from Raji cell nuclei after incorporation of density label from BrdUTP. Logarithmic phase Raji cells were labeled for 7 min with [3H]thymidine (New England Nuclear) at 3 AOCi/ml, made into nuclei by Brij 58 treatment, and allowed to synthesize DNA in a mixture in which BrdUTP (0.03 mM) had been substituted for [3H]dTTP. Samples were washed, lysed, sheared by six passages in a 20-gauge needle, and placed on neutral CsCl gradients as described in Materials and Methods. A260 (0)) and radioactivity (0) by counting of liquid samples in Aquasol (New England Nuclear) were determined for each fraction.

with markers of 1125 and 320 base lengths (data not shown). Similar small material had been seen in experiments in vivo utilizing ['H]thymidine (30). Nuclei from phytohemagglutinin-stimulated lymphocytes were not sufficiently labeled nor obtainable in sufficient quantities to make alkaline sucrose gradient experiments practical. However, it was shown that deoxyribonuclease I treatment of nuclei from phytohemagglutinin-stimulated cells (2 ug/ml, 370, 70 min) did stimulate incorporation, presumably in the form of repair synthesis, equally in the presence (129% of control) and in the absence (138% of control) of ATP. This observation suggested that a simple nuclease-induced repair phenomenon was probably not responsible for incorporation of radioactivity from ['H ]dTTP normally seen in nuclei from phytohemagglutinin-treated cells, but did not rule out the presence of an ATP-dependent deoxyribonuclease activity. Viral and Host DNA Synthesis in the In Vitro System. To pursue the question of replicative compared with repair synthesis in nuclei from Brij-58-treated transformed lymphocytes, experiments to distinguish viral from host DNA synthesis in vitro were performed. The experiments of Hampar et al. (31) suggested that EBV DNA in whole cells is preferentially synthesized at early times in S phase, while most of the host DNA is synthesized at later times. To see if similar results could be obtained in vitro, P3HR-1 cells carrying several hundred copies of the EBV genome per cell (13) amounting to 0.5-1% of the total cell DNA, were partially synchronized by double thymidine block and then placed in culture. Cell samples were removed 15 min, 42 min, and 4.5 hr after removal of the second thymidine block, converted into nuclei by Brij 58 treatment, and then placed in the DNAsynthesizing mixture in vitro. Samples were then washed, lysed, sheared, and sedimented in neutral CsCl gradients. In samples taken at 15 mim after removal of the thymidine block, label was incorporated preferentially into material of about

5,000

BOTTOM

(1975)

1.718 1.700

FRACTIONS

'a

TOP

FIG. 3. Neutral CsCl density gradients of DNA synthesized by nuclei from partially synchronized P3HR-1 cells. P3HR-1 cells were partially synchronized with a double thymidine block (31); after removal of the second block samples were removed from culture at the times indicated, made into nuclei by Brij 58 treatment, and given a 30-min pulse of [3H]dTTP in a DNA synthesis mixture. The nuclei were then treated and sedimented on neutral CsCl as described in Materials and Methods.

density 1.718 [the density of EBV on neutral CsCl (32)] (Fig. 3). In a sample taken at 42 min there was a slight shoulder of radioactive material at density 1.718, but in the 4.5-hr sample only lower density material was present. The latter included material at a density of cellular DNA (1.700) and, in addition, material at an intermediate density. The presence of such intermediate density material has been observed in DNA extracted from cultured lymphocytes (25). In an identical experiment, the EBV-negative T lymphocyte line, Molt, was treated similarly and in parallel with P3HR-1 (Fig. 4). In neutral CsCl gradients, in the presence of a marker of EBV [2H]DNA, nuclei from Molt cells taken 15 min after release of the block showed no incorporation of radioactivity in vitro into material in the region of the marker, while a corresponding sample of P3HR-1 did. Liquid hybridization experiments with EBV complementary RNA also indicated that the denser material on the P3HR-1 gradients in the position of the marker was greatly enriched for EBV DNA. DISCUSSION

DNA synthesis in nuclei prepared from EBV-transformed human lymphocytes by treatment with the detergent Brij 58 was extremely active. These nuclei catalyze DNA synthesis for a much larger period of time than any that have previously been reported. It was of interest to attempt to learn whether this synthesis was due to DNA replication or to a repair process. The synthesis was strongly ATP-dependent. ATP is required in prokaryotic DNA replication in vitro (33) for initiation complex formation. If eukaryotic replication is analogous, the strong ATP requirement (Table 2) suggests that the in vitro system reported here is indeed incorporating

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DNA Synthesis in Lymphocyte Nuclei

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2417

it possible, using the system reported here to determine whether or not it has any role in DNA synthesis or in any other nuclear process. Supported by a grant from the National Institutes of Health (AI-09576).

NUCLEI 10.000-

5,000-

BOTTOM

FRACTIONS

TOP

FIG. 4. Neutral CsCl gradients of DNA synthesized by nuclei from partially synchronized P3HR-1 and Molt cells. P3HR-1 and Molt cells were partially synchronized and treated as described in the legend of Fig. 3. The arrows indicate the location of EBV ['H]DNA, which was added as an internal density marker in each case, and the nuclei were labeled in vitro with [32P] dTTP, as described in Materials and Methods.

radioactivity primarily by a replicative process. This was substantiated by the fact that repair synthesis stimulated by addition of DNase was ATP-independent. Utilization of BrdUTP by the system made possible the demonstration that the material synthesized appeared in stretches of DNA considerably more dense than normal DNA (Fig. 2). Moreover, the existence of small material in the presence of alkaline conditions is consistent with an interpretation of DNA replication by a discontinuous mode (30). This small material is in the approximate size range of intermediates previously reported in discontinuous DNA replication. Additional evidence that replication is probably the predominant mode of incorporation of radioactivity in vitro is contained in Figs. 3 and 4. With nuclei from synchronized cells, label from ['H]dTTP was preferentially incorporated into denser DNA (i.e., viral DNA) at early times and into less dense DNA (i.e., cellular DINA) at later times, consistent with the hybridization data in vivo of Hampar et al. (31). If repair were primarily responsible for the DNA-synthesizing activity observed in this in vitro system, then label should have been incorporated randomly into all types of DNA at all times during S phase. The in vitro system reported here differs in several important respects from a system previously reported by Fridlender et al. (26) for nuclei prepared from normal and phytohemagglutinin-stimulated peripheral human lymphocytes by treatment in a Dounce homogenizer. DNA synthesis in nuclei from Brij-58-treated lymphocytes in vitro resembles other reported systems in its ATP requirement and requirement for absence of calcium (3, 4, 6, 10). In the system of Fridlender et al. (26), calcium was required and ATP was not. They used ['H]dTTP with a specific activity 10- to 20-fold higher than that used here and may have been measuring a much lower level of DNA-synthesizing activity. However, we have no explanation for the differences in properties of the two systems that have been observed. These studies were initiated as part of a study of the nuclear antigen associated with transformation of lymphocytes by Epstein-Barr virus (EBNA) (34, 35). This antigen is in many ways analogous to the T antigen found in cells transformed by papova or adeno viruses. Little is known about the functions of these nuclear antigens. EBNA purification might make

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