Deoxyribonucleases in phytohemagglutinin-stimulated lymphocytes

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Printed in Sweden Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved 0014.4827/79/120365-05$02.00/O

Deoxyribonucleases irk phytohemagglutininstimulated lymphocytes E. JURGEN ZiiLLNER,’ MANFRED REITZ,’ RUDOLF K. ZAHN’ and HANOCH SLOR,2 ‘Physiolog&h-Chemisches Institut der Johannes-GutenbergUniversitiit, Maim, Germany, and 2Department of Human Genetics, Tel Aviv University Medical School, Tel Aviv, Israel

The in situ assay of deoxyribonucleases in DNA-containing polyacrylamide gels following their separation by microdisc electrophoresis was used to determine the deoxyribonuclease pattern of human lymphocytes during stimulation with phytohemagglutinin (PHA). Two additional neutral deoxyribonuclease activities are detectable in stimulated cells, one only active with denatured DNA, the other active with native and denatured DNA as substrate, showing a maximum activity after 36 h and increasing in waves respectively. A group of acid deoxyribonuclease activities also shows a maximum after 36 h of stimulation. A neutral deoxyribonuclease active only with native DNA is missing in stimulated lymphocytes. It is suggested that the acid deoxyribonuclease activities and the neutral deoxyribonuclease active only with denatured DNA are involved in DNA synthesis, whereas the involvement of the neutral deoxyribonuclease active with native and denatured DNA in processing of DNA excreted in stimulated lymphocytes is discussed.

Summary.

There are some reports about deoxyribonucleases (DNases). Loeb et al. [l] described an increase in an alkaline DNase activity paralleled DNA synthesis during PHA stimulation. In lymphocytes of peripheral blood in children an acid lysosomal DNase has been demonstrated by Eschenbath [2] to increase during infections [3]. Leukocytes of the peripheral blood of children suffering from acute leukemia exhibited extremely low activities of this enzyme, but the activity increased rapidly to reach levels of hematologically healthy children following therapeutic treatment [4]. In lymphocytes from normal, Xeroderma pig24-791813

mentosum (X.p.) and heterozygote persons Slor & Lev [5] described an alkaline DNase degrading single-stranded DNA exonucleolytically. Pedrali Noy et al. and Pedrini et al. [6, 71 found a DNase acting on singlestranded DNA and an endonuclease making single-strand breaks in double-stranded DNA in lymphocytes. They determined the levels of these enzymes in cells from normal donors, from X.p. patients and during stimulation with PHA. Both enzyme activities increase in waves during PHA stimulation. In human lymphoblasts Brent [S, 91 showed that there are distinct neutral endonucleases for UV-irradiated and partially depurinated DNA. A serious drawback in the investigations concerning the possible role of all these DNases in biological processes is the fact that the different DNases occur simultaneously. This affords separation prior to activity determination, otherwise an activity increase in one fraction may be compensated by a decrease of another. Using the in situ detection of DNases in DNA containing polyacrylamide gels following their separation by microdisc electrophoresis it is possible to overcome some of these difficulties and to demonstrate four groups of DNase activities simultaneously in human lymphocytes [lo, 11, 121. All activities hydrolyse DNA endonucleolytically. One neutral deoxyribonuclease found in the cytoplasmic fraction prefers native or UVirradiated DNA over denatured DNA as substrate and is a 5’-monoester former. Two groups of acid deoxyribonuclease activities are detectable in the nuclear fraction. Both are 3’-monoester formers. One is active with denatured DNA as well as with Exp

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native DNA; the other shows the same ac- [lo]. Lymphocyte stimulation was performed acto the method of Cooper & Rubin I151. The tivity with native and UV-irradiated DNA, cording cells were cultured in a mixture of 80% Td medium but lower activity with denatured DNA. An 199, 15% heat-inactivated rabbit serum and 500 U/ml 0.5 mg/ml streptomycin. Four ~1 PHA-P alkaline deoxyribonuclease activity, also lo- penicillin, standard solution was added to each culture. The calized in the nucleus, is a 5’-monoester lymphocytes were stimulated at 37°C in a moist atmosphere of 5% CO, for the times indicated. Thyformer, and prefers denatured or UV-irmidine incorporation was measured after adding 2 &i of [3H]TdR/106 cells for 4 h. The determinations of radiated DNA as substrate. DNase activities were performed using the in situ Comparison of the DNase pattern from detection of DNases in DNA-containing polyacrylnormal human lymphocytes with that from amide gels following electrophoretic separation. Beuse the cells were disruoted bv freezine and thawacute lymphocytic leukemic cells, shows fore ing three times. We used a 5 % acrylamidespacer gel, neutral DNase activity to be completely OH 6.7. 0.176 M Tris-H,PO,. and a seoaration ael. i3.4% acrylamide, pH 8.8, 0:177 M Tris:H,SO,. ‘?he missing in leukemic cells of untreated pa- gel electrophoresis was performed at 700 PA/gel for 80 tients, while a group of acid DNase ac- min with the anode at the bottom. The DNA concenin the small pore gel was 0.3 mg native herringtivities is increased. Under medical treat- tration sperm DNA/ml. All operations from gel formation to ment the increment of the acid DNase ac- incubation were carried out at 0°C. A final samole of 8 pi/gel corresponding to an equivalent-of tivities disappeared and the neutral DNase volume 1.6~ 105lymphocytes in spacer gel buffer was applied. activity reappears [ 131. When human lym- After the electrophoretic run the gels with herringDNA were incubated in different incubation phocytes are treated with a mitogenic agent sperm mixtures for 4 h at 37”C, stained with gallocyaninesuch as phytohemagglutinin (PHA) the chromalaune and after destaining with water the opdensity was recorded with a densitometer. The transformation of the small lymphocytes tical spikes of the densitometer tracings were evaluated by into blast cells and the subsequent mitotic comparison with DNase I standard gels. activity are paralleled by a drastic increase in the rate of DNA synthesis. The present Results and Discussion report deals with the change of the DNase Four groups of DNase activities in human pattern of lymphocytes during this stimu- lymphocytes can be demonstrated by the in lation. Beside an increase of acid DNase ac- situ assay of DNases in DNA containing tivities two additional neutral DNases are polyacrylamide gels following their separadetectable, whereas another neutral DNase tion by microdisc electrophoresis [ 10, 111. These activities had been characterized is missing in stimulated lymphocytes. with respect to their electrophoretic moMaterials and Methods bility, optimal incubation conditions, substrate preference, mode of action and subMaterials were obtained as follows: Ronpacon 75% from Cilag Chemie GmbH, Alsbach; Thrombovetren cellular localization [lo, 121. During stimu(4000 IE heparinR ml) from Promota, Hamburg; Ficoll from Pharmacia Fine Chemicals AB. Uousala: tissue lation with PHA some alterations of the culture media from Serva, Heidelberg;‘PHA-P from DNase activities in lymphocytes can be obDifco Laboratories, Detroit, Mich.; Gallocyanine from served (fig. 1). The acid DNase activities B Fluka AG; Chromalaune from Merck, Darmstadt; DNase I (EC 3.1.4.5) 2000 U/mg from Worthington, increase with a maximum after 36 h (fig. Freehold, N.J.; herring-sperm DNA, prepared ac1b), and the neutral DNase A disappears cording to Zahn et al. [14], was a gift from H. Mack, Illertissen; [methyl-3H]thymidine (spec. act. 5.0 Ci/ completely after 12 h (fig. If). A neutral mmol) from the Radiochemical Centre, Amersham. All other reagents were of the highest analytical grade as DNase PZ only detectable with denatured supplied by Serva, Heidelberg, and Merck, Darmstadt. DNA as substrate and with the same elecHeat-denatured DNA was prepared by heating native herring-sperm DNA for 10 min at 100°C in a boiling trophoretic mobility as the alkaline DNase water bath and chilling in ice. Lymphocytes were iso- D but not detectable at alkaline incubation lated from peripheral blood of normal donors and separated in a Ficoll-Isopaque gradient as described conditions also increases (fig. Id). After a Exp Cell Res 123 (1979)

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decreasing optical density. Densitometer tracings of DNA-containing polyacrylamide gels after electrophoresis of freeze-thawed PHA-stimulated lymphocytes. The gels contained 0.3 mg native (a, b, e, fl or denatured (c, d) herringsperm DNA/ml in 13.4% acrylamide separation gel, pH 8.8. On each gel 8 ~1 of an equivalent of 20X106 lymphocytes/ml spacer gel buffer was applied. The cells were cultivated for 36 h as described in Materials and Methods, a, c, e, without; b, d,f, with PHA. After electrophoresis the gels were incubated for 4 h; a, b, in 0.1 M Na acetate pH 5.0, 1.5 mM EDTA; c, d, e, f, in 0.1 M Tris-HCl, pH 7.5, 10 mM MgCl,. The letters P, and PZ refer to the different groups of DNase activities only detectable in PHA-stimulated lymphocytes.

Fig. 1. Ordinate:

Fig. 2. Abscissa: duration of culture (hours); ordinate: (a) % of maximum; (b) x lo4 cpm.

Variations in the activity of two neutral DNases during PHA stimulation. Cultures of human lymphocytes were treated with PHA for the indicated times. (a) The cells were collected and assayed for DNase activity by the in situ detection of DNases in DNAcontaining polyacrylamide gels. +-+, Neutral DNase P,; O-O, neutral DNase PI; (see Materials and Methods and fig. 1); (b) incorporation of [3H]TdR.

phoretic mobility, increases in waves (fig. Id, f, 2~). These two DNases must be separated before activity determination, maximum at 36 h this activity shows a gradual decline and is no more demon- otherwise the different behavior during strable after 84 h (fig. 2~). An additional stimulation cannot be observed, since both neutral DNase PI, active with native or de- are active under the same incubation connatured DNA as substrate and discernable ditions with native DNA as substrate. These data show that the DNase pattern from the DNase A active under the same incubation conditions by its different electro- in PHA stimulated lymphocytes at the time

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of maximum [3H]TdR incorporation (fig. 2b) corresponds to the DNase pattern of acute lymphocytic leukemic (ALL) cells with respect to the increased fast electrophoretically migrating DNase activities B and the loss of the neutral DNase A [13]. A high activity of a DNase, corresponding to the activity P, with a clear maximum at the highest rate of DNA synthesis (fig. 2a, b), is also found in ALL cells. All this supports the hypothesis that both groups of enzymes, the fast migrating acid DNase activities B and the DNase Pz may be involved in DNA synthesis. The neutral DNase A, missing in ALL cells [ 121also disappears during stimulation and may be involved in specific functions of peripheral lymphocytes. This is supported by the fact that the reappearance of sufficient functional intact lymphocytes in the peripheral blood during treatment of ALL patients is accompanied by a demonstrable neutral DNase A [12]. DNases in lymphocytes may be involved in excretion of DNA. Rogers [16] showed that PHA induces lymphocytes to replicate selectively several copies of a limited portion of their genome, copies which are then excreted into the culture medium. As determined by reassociation kinetics analysis, a high molecular weight DNA fraction from the Hirt supernatant contains sequences found in excreted DNA. This DNA may represent an intermediate formed prior to release of excreted sequences form the cells and DNases, increasing during stimulation, can be involved in the processing of this DNA to the low molecular weight DNA excreted into the medium. To evaluate this hypothesis more data about the intracellular location and the substrate specificity of this endonuclease in respect to the excreted DNA are necessary. The DNase P, shows no direct correlation to DNA synthesis (fig. 2a, b). Exp Cd Rrs I23 (1979)

No DNase activity described here increasing at the time of high DNA synthesis correlates to the DNase described by Loeb et al. [ 11, because this activity differs in respect to incubation conditions and substrate preference. Also Pedraldi Noy et al. and Pedrini et al. [6, 71 showed DNases increasing during PHA stimulation of human lymphocytes. The single-stranded DNA DNase, like DNA polymerase activity, showed a peak at maximum of [3H]TdR incorporation and a second one at about 9 days when DNA synthesis rate is not showing any increase. The endonuclease also increased reaching a maximum at a time when DNA synthesis is down to the background level or decreasing. From the data available, it could be proposed that the singlestrand DNA DNase might correspond to the DNase PZ and the endonuclease to P,. The fact that the change of the enzyme activities is slightly different need not be a contradiction. In the DNase test system with crude cell extracts without separation of different enzyme activities before assay used by these authors, one enzyme can simulate to a lesser extent an activity of a different group. So has the single-stranded DNA DNase also about 15% activity with native DNA [7]. Using the in situ assay of DNases in DNA-containing polyacrylamide gels following their separation by disc electrophoresis this can be avoided. The increase of the acid DNase activities B is also in accordance with reports about elevated acid DNase activities during rat liver carcinogenesis induced by various carcinogens. Thus, Schneider et al. [17], Brody [ 181, Schulze [19] and Tempel & Hollatz [20] correlated this observation with the increased DNA synthesis during this process. Using different mitogens for stimulation of rat lymphocytes Slor et al. [21] also described a unique DNase which seems to be

Preliminary notes similar to the neutral DNase P, described here. We wish to thank Mrs R. Nerhbal3 for technical assistance. This work has been supported by the Stiftung Volkswagenwerk.

References 1. Loeb, L A, Edwald, J L & Agarwal, S S, Cancer

res 30 (1970) 2514. Eschenbach, C, Histochemie 24 (1970) 85. - Klin Wschr 49 (1971) 949. - Ibid 49 (1971) 959. Slor, H & Lev, T, Biochim biophys acta 312 (1973) 637. 6. Pedrini, A M, Dalpra, L, Ciarrocchi, G, Pedrali Noy, G C F, Spadari, S, Nuzzo, F & Falaschi, A, Nucleic acids res 1 (1974) 193. 7. Pedrali Noy, G C F, Dalpra, L, Pedrini, A M, Ciarrocchi, G, Giulotto, E, Nuzzo, F & Falschi, A, Nucleic acids res 1 (1974) 1183. 8. Brent. T P. Biochim bionhvs acta 407 (1975) 191. 9. - Nucleic’acids res 4 ([97?‘) 2445. 10. Zollner, E J, Helm, W, Zahn, R K, Beck, J & Reitz, M, Nucleic acids res 1 (1974) 1049. 11. Zollner. E J. Sterner. H. Breter. H J & Zahn. R K, Z Naturforsch 30: (1975) 781. 12. Zollner, E J, Weinblum, D, Obermeier, J & Zahn, R K, Exp cell res 99 (1976) 185. 13. Zollner, E J, Beck, J-D, Lemmel, E M & Zahn, R K, Cancer lett 1 (1975) 119. 14. Zahn, R K, Tiesler, E, Kleinschmidt, A K & Lang, D, Biochem Z 336 (1962) 281. 1.5. Cooper, H L & Rubin, A D, Blood 25 (1965) 1014. 16. Rogers, J C, Proc natl acad sci US 73 (1976) 3211. 17. Schneider, W C, Hojeboom, G H, Shelton, E & Striebich. M J. Cancer res 13 (1953) 285. 18. Brody, S; Nature 182(1958) 1386. ’ 19. Schulze. N B. Z Krebsforsch 79 (1973) 241. 20. Tempel, K & Hollatz, R, Z Krebsforsch 79 (1973) 267. 21. Slor, H, Leve-Sobe, T & Kalina, M, Exp cell res 101 (1976) 416. 2. 3. 4. 5.

Received August 23, 1978 Revised version received July 3, 1979 Accepted July 11, 1979

Printed in Sweden Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/79/120369-06$02.00/O

Do clusters of replication mammalian cells exist?

units in the

YU B. YUROV, Institute demy of Medical

of Medical Genetics, AcaSciences, Moscow 115478, USSR

Evidence against the existence of clusters of synchronously operating units in human and Chi-

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nese hamster cells has been presented by means of the DNA fiber autoradiography. The analysis of DNA molecule autoradiographs obtained by different labelling protocols shows that the appearance of tandemly arrayed units may be an artifact associated with fibre autoradiography. The data obtained indicate that the method of measuring centre-to-centre distances previously used to estimate replication unit size is not reliable. An attempt to define the heterogeneity of replication unit size shows that the distances between the centres of adjacent replication units vary from 5 to 500 pm, average value 120pm.

The first attempt to determine directly the size of replication units in mammalian cells was made by Huberman & Riggs [ 11. They have presented evidence for a clustering distribution of active replication units on a single DNA molecule and proposed measuring the size of replicons as distances between adjacent initiation points. Experiments of this kind have shown that in mammalian cells the distances between initiation points of replication vary from 10 to 100 pm (average about 30-60 pm) [l-3]. Recently, evidence has been presented [4] for about 4 pm replication units in CHO cells. These data were based on results by studying DNA fibre autoradiographs from CHO cells, which were highly synchronized at the beginning of S phase by mitotic selection and blocking of DNA synthesis with fluorodeoxyuridine (FdU). The size of the replication units measured as center-tocenter distances of replicating fragments after a pulse of 4 min is about 4 pm over a large part of chromosomal DNA. As the mean value of replication unit sizes in CHO cells is about 30-60 pm [ 11,these data were interpreted “as evidence for regularly spaced initiation sites which are available in CHO cells, even though only one in 10-15 of these may be utilized for the initiation of each cycle under normal growth conditions in the cultures” [4]. All of these data on replicon sizes are based on the assumption that when arranged in clusters and simultaneously repliE-rl, CdRes

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