Quiescent human peripheral blood lymphocytes do not contain a sizable amount of preexistent DNA single-strand breaks

Experimental Cell Research 180 (1989) 569-573

SHORT NOTE Quiescent Human Peripheral Blood Lymphocytes Do Not Contain a Sizable Amount of Preexistent DNA Single-Strand Breaks

MICHAEL E. T. I. BOERRIGTER? ERIK MULLAART,* GOVERT P. VAN DER SCHANS,‘l’ and JAN VIJG* *TN0 Institute for Experimental Gerontology, P.O. Box 5815, 2280 IIV Rijswijk, The Netherlands, and TTNO Medical Biological Laboratoqs, P.O. Box 45, 2280 AA Rijswijk, The Netherlands

Sedimentation of nucleoids through neutral sucrose density gradients has shown that nucleoids isolated from phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes (PBL) sediment faster than nucleoids derived from quiescent lymphocytes, which was attributed to rejoining of DNA single-strand breaks (SSB) present in the resting cells (A. P. Johnstone, and G. T. Williams (1982) Nuture (London) 300, 368). We isolated PBL from donors and determined the amount of SSB in nonradiolabeled, untreated resting and PHA-stimulated cells by applying the alkaline filter elution technique. Calibration was based on dose-dependent induction of SSB by @‘Cp/-radiation. Quiescent cells did not contain a sizable amount of SSB. Mitogen-stimulated cells showed equally low amounts of SSB per cell. The present study indicates that the interpretation of the results obtained with the nucleoid sedimentation technique concerning the supposed rejoining of SSB in PHA-stimulated human lymphocytes is incorrect. Other. equahy sensitive. techniques such as alkaline filter elution appear to be preferable for studies on DNA damage and repair. Q 19x9 Academic Press, Inc.

Activation of human peripheral blood lymphocytes (PBL) is thought to be accompanied by the joining of preexistent DNA breaks in quiescent PBL 11, 21. Analysis of the rate of sedimentation of nucleoids, i.e., supercoiled DNA that has lost most of the nuclear protein, showed that such nucleoids from phytohemagglutinin (PHA)-stimulated PBL sedimented faster through the neutral sucrose density gradients than those derived from quiescent lymphocytes [I]. This increased sedimentation rate was interpreted as the effect of rejoining of singlestrand breaks (SSB) present in the DNA of resting cells. The change in sedimentation rate was smaller when inhibitors of ADP-ribosyltransferase (ADPRT) were included in the culture medium of activated lymphocytes [ 11. Because ADPRT is involved in increasing the activity of DNA ligase II 133this effect was thought to be indicative of an obligatory role of DNA ligation during lymphocyte activation

[Il. Nucleoid sedimentation has been used extensively for studying the effect of radiation or carcinogen treatment on cellular DNA [4-71. As compared to nucleoid sedimentation, the technique of alkaline elution as developed by Kohn et (11.[8] may measure SSB more directly, since it is not based on changes in DNA ’ To whom reprint requests should be addressed.

570 Short note

l0

L IO

5 eiution

I

, 15

time (h)

Fig. 1. Alkaline elution of DNA through membrane filters. Elution curves for freshly isolated P either untreated or exposed to various doses of y-radiation. Elution was plotted as the log percentage of DNA remaining on the filter as a function of time. The initial slopes of the various curves are 0 Gy, 0.015; 1 Gy, 0.08; 2 Gy, 0.13; 4 Gy, 0.22; 8 Gy, 0.50.

tertiary structure but on the degree of unwinding under specified conditions, which is determined by the number of SSB present. This work demonstrates that according to alkaline elution results ~~st~m~lated PBL do not contain a sizable number of SS , or alkali-labile sites, at the ti isolation. Stimulation with PHA did not affect the level of detectable s results are discussed in relation to the observed changes in the nucleoi tation rate of stimulated PBL as compared to that of restmg cells. Materials and Methods Cell isolation and culture. Human peripheral blood was collected from healthy volunteers not taking medication. Lymphocytes were isolated using Ficoli-Paque (Pharmacia, Sweden) gradients 191, washed twice in RPM1 1640 medium (Flow Laboratories, UK) plus 2% fetal calf serum (FCS). ,411 steps were performed at 4°C. Lymphocytes that were stimulated with PHA (Wellcome Foundation Ltd., UK) were resuspended at a final concentration of 2~ lo6 cells/ml in RPM1 1640 medium plus 10% FCS supplemented with 2 mM glutamine and antibiotics. Stimulation with PHA (lo &mi) was for 3 days at 37°C. The activation status of mitogen-stimulated PBL was verified by measuring he incorporation of [methyl-3H]thymidine (25 Ci/mmol, 10 @X/ml; Amersharn, UK) in control and PHAstimulated cultures over the last 18-h period of culture. Defection ofDIVA strand breaks. The technique of alkaline filter elution was used to measure SSB as previously described [lo]. Cells were resuspended in cold phosphate-buffered saline (PBS) at a concentration of 2x IO6 cells/ml; 0.8-l x lo6 cells were applied per filter. Loading and iysing of the cells, as well as the elution of the DNA, were performed under subdued lighting in order to minimize artificial induction of strand breaks [ll]. After the addition of Hoechst 33258, DNA in each fraction was quantitated spectrofluorometrically as described [lo]. The elution result was plotted as the log

Short note 571 TABLE 1 SSB and alkali-labile sites in human white blood cells

Quiescent” Quiescentb QuiescenF PHA-stimulatedd

It

x

16 9 6 3

345 456 358 519

SD

Range

114 163 115 16.5

148-476 226-691 14W36 378-700

Note. The number of SSB plus alkali-labile sites in DNA was determined by alkaline elution in freshly isolated PBL. Lymphocytes from donors N18 and NO1 were analyzed repeatedly over a period of 1 year, with intervals of 5 weeks. a Different donors; one blood sample per person. b Donor N18. ’ Donor NOl. d Lymphocytes from three different donors were stimulated with PHA (10 ugiml; 37°C) and analyzed at the third day of stimulation. Activation of PBL was 40- to 50-fold. Control cultures without mitogen incorporated (mean f SD) 1200 (+558) cpm, whereas PHA-stimulated cells incorporated 61,149 (& 2563) cpm.

percentage of DNA remaining on the filter as a function of time. Mean slopes of the linear part of the elution curves were used to calculate the number of SSB plus alkali-labile sites by calibration with mean elution curves of cells exposed to 4 Gy 60Co-y-radiation (Gamma-cell 100, Atomic Energy of Canada Ltd.; dose rate 6 Gyimin), which were assayed in the same experiment. At 4-Gy y-radiation approximately 4000 SSB per diploid genome is introduced 1121.The amount of SSB in unirradiated cells was obtained by using the formula 4000x

slope of the elution curve of unirradiated cells slope of the elution curve of 4-Gy y-irradiated cells

In all experiments the mean slope was based on at least triplicate determinations.

Results and Discussion The alkaline elution technique was applied to untreated PBL and to cells exposed to various doses of %Zo-y-radiation. Figure 1 shows typical elution profiles. On the semilogarithmic plots the slope of the initial part of the curve increases linearly with increasing dose. This indicates that the rate of elution (slope) is proportional to the number of SSB induced by y-radiation within the range O-8 Gy. It is clear that under these conditions low numbers of SSB or sites in DNA that turn into SSB in alkali can readily be quantitated. With respect to unirradiated lymphocytes, almost all the DNA is retained on the filter which shows that only a few SSB are present or formed. The absence of a sizable amount of SSB in freshly isolated quiescent PBL was confirmed with cell samples from other donors. The amount of SSB per cell (mean + SD) was 345 (+ 114) (Table 1). The observed variation in the level of SSB could result from methodological factors or reflect a biological variation, that is, a small but different level of preexistent SSB in resting PBL from different donors. To determine the methodological variation in the amount of SSB detected in

572

Short note

quiescent PBL, cells were isolated from blood samples collected repeate two donors, which were analyzed for SSB with the alkaline elutio eve1of SSB per cell was 456 (2163) for donor Nl8 and 358 (tl NO1 (Table 1). The coefficient of variation ranged from 10 to 5 isolated from different donors as compared to I-50% and IQ-59 cytes obtained from sequential blood samples of donor Nl8 and respectively. The difference between the coefficients of variation donors on the one hand and for the same donor on the other is small, ~~d~~~t~~g that the variation in the sample populations is about the same. This suggests that the observed variation in the level of SSB stems from rnetbodo~o~~~a~ y, possibly due to background breaks induced during isolation of th n contrast to the supposed disappearance of SS stimulation, as concluded from the increased ra of sedimentation of n [I], we found no differences in the amount of S after stimulation of PHA as compared to freshly isolated cells. The level of SS (+ 1651,not significantly different from the values for restin The increased rate of sedimentation in sucrose density gr -stimulated PBL has been interpreted as the rejoining of of resting lymphocytes. However, in view of t results obtained with alkaline elution, this interpretation since the nucleoid sedimentation is less irect with regard to e detection of S than alkaline unwinding or alkaline sucrose gradient sedime discrepancy may be relevant in this respect: human skin blasts show comparable levels of repair replication after ultraviolet light-inradiation, but they differ in the rate at which their nucleoids recover normal sedimentation behavior [ 131. Considering the structure of the nucleoid and the fact that quiescent P not contain a high level of preexistent strand breaks, the observed differences m sedimentation rate of nucleoids isolated from quiescent a lymphocytes may be explained by differences in compactness this regard it it not inconceivable that nucleoids isolated from have a more compact supercoiled structure resulting in an entation though neutral sucrose gradients. Changes in the DNA-bound ligands in viva such as RNA and protein may affe density of nucleoid DNA [ 141. Nucleoid sedimentation is a very sensitive method to detect small changes in the number of SSB [5]. However, alterations in. the rate of nucleoid sedi~~~~at~~~ can arise not only from changes in the amount of strand breaks but also from changes in DNA supercoiling. Given the high number of artifacts [I§] and the sometimes difficult interpretation of the results [13, 151 it is preferable to use other, equally sensitive, techniques such as the alkaline filter elution [I direct determinations of DNA damage and study of its repair. This work was supported by grants from Senetek PLC and the Dutch Ministry of Welfare and Health Affairs. We thank Dr. F. Berends for critically reading the manuscript and 34. M. Boermans for preparing the figure.

Short note 573 References 1. Johnstone, A. P., and W~li~s, G. T. (1982) Nature(~o~~o~) 300, 368-370. 2. Carson, D. A., Seto, S., Wasson, B., and Carrera, C. J. (1986) Exp. Cell Rex. 164, 273-281. 3. Creissen, D., and Shah, S. (1982) Nature (London) 296, 271-272. 4. Cook, P. R., and Braze& I. A. (1975) J. Cell Sci. 19, 261-279. 5. Cook, P. R., and Braze& I. A. (1976) Nature (London) 263, 679-682. 6. Farzaneh, I?., Zahn, R., Brill, D., and Shah, S. (1982) Nature (London) 300, 362-366. 7. Romagna, F., Kulkami, M. S., and Anderson, M. W. (1985) B&him. Biophys. Acta 127, 56-62. 8. Kohn, K. W., Erickson, L. C., Ewig, R. A. G., and Friedman, C. A. (1976) Biochemistry 15, 4629-4637. 9. Boyum, A. (1968) &and. J. Clin. Lab. Invest. 21, 77-89.

10. Mullaart, E., Boerrigter, M. E. T. I., Brouwer, A., Berends, F., and Vijg, J. (1988) Me&. Age& Dev., in press. 11. Bradley, M. O., Erickson, L. C., and Kohn, K. W. (1978) Biochim. Biophys. Acta 520, 1l-20. 12. Schans, G. P. van der, Centen, H. B., and Lohman, P. H. M. (1982) in Progress in Mutation Research (Natarajan, A. T., Obe, G., and Ahmann, H., Eds.), Vol. 4, pp. 285-299, Eisevier Biomedical, New York. 13. Charles, W. C., and Cleaver, J. E. (1982) Biochem. Bio~~ys. Res. Common. 107, 2.50-257. 14. Cook, P. R., and Brazell, I. A. (1976) f. Cell Sci. 22, 303-324. 15. Weniger, P. (1982) in Progress in Mutation Research (Natarajan, A. T., Obe, G., and Altmann, II., Eds.), Vol. 4, pp. 261-265, Elsevier Biomedical, New York. Received July 11, 1988 Revised version received September 12, 1988

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