May 23, 1985 - a separate series of experiments, demecolcine (Colcemid;. Ciba-Geigy, Basel, Switzerland) was added to the 72-h lymphocyte cultures for 6 h ...
MOLECULAR AND CELLULAR BIOLOGY, Nov. 1985. p. 3270-3273 0270-7306/85/113270-04$P2.00/0 Copyright © 1985, American Society for Microbiology
Vol. 5, No. 11
Binding of Anti-Z-DNA Antibodies in Quiescent and Activated Lymphocytes: Relationship to Cell Cycle Progression and Chromatin Changes LISA STAIANO-COICO,l* B. DAVID STOLLAR,2 ZBIGNIEW DARZYNKIEWICZ,3 REGINA DUTKOWSKI,4 AND MARC E. WEKSLER4 Departments of Surgery' and Medicine,4 Cornell University Medical College, New Yort, New York 10021; Deprtment of Investigative Cytology, Sloan-Kettering Instituite, Walker Labor-atories, Rve, Newii York 105803; and Department of Biochemistry and Pharmacology, Tufts University Medical School, Boston, Massachuisetts, 02111' Received 23 May 1985/Accepted 19 August 1985
Although regions of DNA reacting with anti-Z-DNA antibodies have been identified in the polytene chromosomes of Drosphila spp. and the metaphase chromosomes from a number of different mammalian species, the biological role of this DNA is unknown. Flow cytometry was used in the present studies to quantitate the binding of anti-Z-DNA antibodies in quiescent and activated human peripheral blood lymphocytes; the antibody binding was then correlated with cell cycle phase. The data show that quiescent (GO or GIQ) lymphocytes are heterogeneous with respect to their reaction with anti-Z-DNA antibodies. The transition from quiescence (G1Q) into the cell cycle (GI), which involves decondensation of chromatin, did not result in any significant change in binding of these antibodies. In contrast, progression of cells from GI through S and G2 is correlated with a '27% decrease in anti-Z-DNA antibody reactivity relative to total DNA content. No significant change was observed during the transition from G2 to mitosis (M).
Left-handed Z-DNA has been shown to exist in crystals and can be induced in polynucleotide solutions in certain ionic environments and in supercoiled DNA (9, 15, 16, 20). This and other stabilized forms of Z-DNA are immunogenic, and antibodies specific for Z-DNA have been raised by a number of investigators (10, 11, 13). The anti-Z-DNA antibodies have then been used to identify areas within the DNA molecule that can assume the left-handed form in polytene chromosomes of insects (2, 14) and metaphase chromosomes (19) of mammalian cells by in situ immunofluorescence. In the present study we have used multiparameter flow cytometry to measure the binding of anti-Z-DNA antibodies within the nuclei of quiescent and mitogen-stimulated human peripheral blood lymphocytes in relation to total DNA content. Thus, anti-Z-DNA antibody binding could be measured and compared in cells at different stages of the cell cycle and with various degrees of chromatin condensation inasmuch as chromatin of these cells undergoes decondensation during their transition from quiescence to the cell cycle.
48, 72, and 96 h after the addition of PHA, the cells were harvested by centrifugation and fixed in ice-cold 70% ethanol. Prior to antibody staining, fixed cells were centrifuged (150 x g; 5 min) and suspended in HBSS. In one series of experiments, cells were preincubated for 5 min in either 0.08 N HCI (pH 1.65) or 0.1 M phosphate-citrate buffer (pH 2.60) at room temperature prior to reacting with the anti-Z-DNA antibodies. Goat (polyclonal) or murine (monoclonal) antiZ-DNA antibodies were prepared and purified as previously described (11, 18). The cell suspensions were reneutralized and centrifuged as above, and the cell pellets were resuspended in 25 [L of either goat or murine anti-Z-DNA antibodies (1 mg/ml protein), normal goat serum, or normal mouse serum and incubated for 30 min. The cells were washed twice and collected by centrifugation in HBSS as described above, and the cell pellets were suspended in 50 ,ul of a 1:40 dilution of either fluorescein isothiocyanateconjugated rabbit anti-goat immunoglobulin or goat antimouse immunoglobulin antisera (Cappel Laboratories, Cochranville, Pa.). The cells were incubated in the presence of fluorescein isothiocyanate-conjugated antibody to immunoglobulin for 30 min, then washed twice in HBSS, incubated with RNase A (Sigma Chemical Co., St. Louis, Mo.), and counterstained with propidium iodide (25 ,ug/ml final concentration; Polysciences, Warrington, Pa.) as described previously (3). Anti-Z-DNA antibody binding was competitively inhibited specifically by brominated poly(dG-dC) in Z conformation and not by other nucleotides; furthermore, immunofluorescence was abolished by preincubation in DNase I but not RNase A. To estimate the proportion of cells in G1Q, S, G2, and M, the method of simultaneous staining of RNA and DNA with acridine orange (Polysciences, Warrington, Pa.) was used as described previously (8). Green (515 to 560 nm) and red (620 nm) of individual cells were measured in an Ortho 50H cell sorter interfaced to an Ortho 2150 computer (Ortho Diagnostic Instruments, Westwood, Mass.). The correlation be-
MATERIALS AND METHODS Blood from healthy donors was diluted with an equal volume of Hanks balanced salt solution (HBSS; GIBCO Laboratories, Grand Island, N.Y.), layered over Ficollisopaque (Lymphoprep; Nyegaard Chemical Co., Oslo, Norway), and centrifuged (400 x g) for 20 min at room temperature. The mononuclear cells removed from the interphase was rinsed with HBSS, suspended (106 cells per ml) in RPMI 1640 containing 15% fetal bovine serum, 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 ,ug/ml), and cultured in the absence or presence of 5 p.g of purified phytohemagglutinin (PHA; Burroughs Wellcome Co., Research Triangle Park, N.C.) per ml in a 5% C02-95% humidified air environment for up to 96 h. Before and at 24,
*
Corresponding author. 3270
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TABLE 1. Cell cycle progression of PHA-stimulated lymphocytes" Time in
Cell phase
presence of PHA (h)
G1Q
GI
S
G2 plus M
0 24 48 72
99.3 75.9 63.2 20.6
0.7 22.7 20.4 38.8
0.0 0.7 7.6 25.9
0.0 0.7 8.8 14.7
"Peripheral blood mononuclear cells were cultured in the presence of PHA; 0, 24, 48, and 72 h after the addition of PHA, portions were withdrawn from cultures and stained with acridine orange, and their cell cycle distribution was determined by flow cytometry as described previously (4). Parallel samples were investigated with respect to anti-Z-DNA antibody binding (see Table 2).
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antibodies (Table 3). An increase in fluorescence intensity was observed when lymphocytes were preincubated at either pH 2.60 or 1.65. Whereas 88% of the cells had detectable amounts of Z-DNA after pretreatment at physiologic pH, 99% of the cells pretreated at low pH had detectable amounts of Z-DNA. Thus, it appears that pretreatment at low pHs increased the binding of anti-Z-DNA antibodies by either increasing the accessibility of the antibodies to sites of DNA which were in the Z conformation or stabilizing those sites which were predisposed for the Z conformation. The increase in fluorescence intensity was observed equally throughout all phases of the cell cycle. In agreement with our previous observations, the amount of propidium iodide
tween immunofluorescence and cell cycle phase was determined as previously described (3). The significance of experimental results was determined by the paired Student t test.
RESULTS The proportion of cells in GO (G1Q), G1, S, and G2 plus M was estimated by flow cytometric analysis of correlated measurements of differentially stained RNA and DNA (8). Prior to the addition of PHA, more than 99% of all lymphocytes were in the G1Q phase of the cell cycle. Within 24 h after the addition of PHA, 23% of the lymphocytes had entered G1, as discriminated by increased RNA content (Table 1; reference 8). Within 48 h, 63% of the lymphocytes had been activated, and a significant number were already in S (7.6%) and G2 plus M (8.8%) phases of the cell cycle. After 72 h in culture with PHA, the time of peak [3H]thymidine incorporation, only 21% of the cells remained in the G1Q compartment, and a maximal number of cells was observed in S phase. Correlated measurements of cellular DNA content versus anti-Z-DNA antibody immunofluorescence are shown in Fig. 1. It is apparent that quiescent lymphocytes are heterogeneous with respect to anti-Z-DNA antibody binding (Fig. 1B); approximately 16% of the cells had no detectable Z-DNA. Although activated lymphocytes (G1) from cultures exposed to PHA for 24 h could be discriminated on the basis of increased cellular RNA content (8), they could not be distinguished on the basis of anti-Z-DNA antibody binding (Fig. 1C). The fluorescence intensities of G1 comnpared with those of S and G2 plus M lymphocytes in cultures from 14 separate donors are shown in Table 2. There was an increase in the amount of Z-DNA detected when the cells entered S phase (P < 0.005), and there was a further increase when they entered the G2 plus M phases of the cell cycle. However, when DNA content per cell was taken into account, the actual amount of detectable Z-DNA was decreased by 27% in the G2 plus M phases of the cell cycle. In a separate series of experiments, demecolcine (Colcemid; Ciba-Geigy, Basel, Switzerland) was added to the 72-h lymphocyte cultures for 6 h prior to harvest. While the presence of demecolcine increased the number of metaphase cells in the G2 plus M population from 14.7 to 28.1%, it did not significantly alter the relative mean fluorescence intensity of this population. All of the above experiments were performed on cells which had been treated with anti-Z-DNA antibodies at physiologic pHs. It has been reported that brief preincubation of cells at low pH enhances the binding of anti-Z-DNA antibodies (17). We performed a series of experiments in which lymphocytes were pretreated with low-pH buffers, neutralized, and then exposed to the primary anti-Z-DNA
W.'A
FIG. 1. Correlation of anti-Z-DNA antibody binding (log scale) and cell cycle progression (linear scale) in lymphocyte cultures incubated in the presence of PHA for 0 (A and B), 24 (C), 48 (D), and 72 (E) h. Panel A represents lymphocytes incubated in the presence of normal goat serum, followed by rabbit anti-goat immunoglobulin; it is evident that the nonspecific fluorescence is minimal. FITC, Fluorescein isothiocyanate; P1, propidium iodide.
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MOL. CELL. BIOL.
bound to DNA was also increased after pretreatment with acid (7). Exposure of cells to normal goat immunoglobulin G did not lead to immunofluorescence. Preincubation of goat antiZ-DNA with brominated poly(dG-dC) eliminated the staining, whereas preincubation with native or denatured calf thymus DNA or poly(I)-poly(C) did not. Purified rnurine monoclonal anti-Z-DNA yielded the same results as polyclonal goat serum, whereas normal mouse immunoglobulin G caused no immunofluorescent staining. DISCUSSION The localization in situ and the function of Z-DNA are to a large extent unknown. DNA reactive with anti-Z-DNA antibodies can be found in the band or interband regions of chromosomes, depending on the procedure used to fix a specimen (2, 11, 12). The possible role(s) of chromatin proteins, including modifications by acetylation or phosphorylation, on the induction or stabilization of the Z conformation is not known. In addition, there have been no comparisons betweeh the chromatin of quiescent cells and cycling cells (i.e., mitogen-activated lymphocytes). To approach some of these questions, we used flow cytometry to examine the binding of anti-Z-DNA antibodies by human peripheral blood mononuclear cells cultured with PHA. Lymphocytes from such cultures allowed a comparison of quiescent cells (G1Q) with cells progressing through the cell cycle (8). In addition, G1Q cells which contained predominantly condensed chromatin could be compared with their cycling counterparts which contained predominantly dispersed chromatin. The majority of the cells studied bound the anti-Z-DNA antibodies extensively enough to be detected and measured by flow cytometry. The binding appears to be specific for sites of DNA which either have Z conformation or a predisposition to assume Z conformation under the conditions of ethanol fixation. The staining was abolished by DNase I. An increase in antibody binding and propidium iodide fluorescence was observed in cells pretreated with acids and was TABLE 2. Z-DNA antibody binding to lymphocytes in 72-h PHA cultures" Cell cycle phase
GtQ-G1 S G, plus M G2 plus M with demecolcine
Fluorescence intensity Per DNA Per cell unit
1.00 1.26 ± 0.20 (P < 0.005) 1.46 ± 0.25 (P < 0.005) 1.37
1.00
0.84 0.73 0.69
" PHA-stimulated cells were stained with anti-Z-DNA antibodies and counterstained with propidium iodide for total DNA content. The mean green fluorescence intensities (anti-Z-DNA antibody binding) of cells within each of the phases of the cell cycle were measured by flow cytometry; 104 cells were counted per sample. The mean fluorescence intensities of GQ(-G, cells were normalized to 1.00 in each sample, and the mean fluorescence intensities of S and G2 plus M cells were expressed relative to those of GQ.-Gl cells. Fourteen different cultures from different individuals were analyzed, and the mean values ± standard deviation are shown (P values indicate differences with respect to the G1Q-G1 population). In addition, cells were analyzed from a culture treated with demecolcine for 6 h prior to harvest. (G2 plus M with demecolcine). Although demecolcine treatment resulted in an increase in the proportion of G2 plus M cells from 14.7 to 28.1%, indicating a severalfold enrichment in metaphase cells, the mean fluorescence intensity was not significantly changed relative to the G2 plus M population in asynchronous cultures. Taking into account DNA content per cell, a decrease in anti-Z-DNA antibody binding occurred during cell cycle progression.
TABLE 3. Increased binding of anti-Z-DNA antibodies after pretreatment with acids" Cell pretreatment (pH)
Mean fluorescence intensity
7.20 . .1.00 2.60 .1.33 ± 0.09 (P < 0.02) 1.65 .1.50 ± 0.09 (P < 0.001) "' Prior to anti-Z-DNA antibody binding, cells were preincubated for 5 min at pH 7.20. 2.60, or 1.65. The mean green fluorescence of the G,Q-G, population at pH 7.20 was normalized to 1.00 in each sample, and the fluorescence intensities of the G,Q-G, populations of cells pretreated at either pH 2.60 or 1.65 were related to this value. The experiment was performed on peripheral blood lymphocytes from 10 different donors (mean ± standard error of the mean); P values relate to an increase in fluorescence in relation to pH 7.20.
especially prominent after pretreatment at pH 1.65. At that pH, histones and certain nonhistone proteins can be extracted from nuclei, and DNA becomes significantly more accessible to a variety of fluorochromes, both intercalating dyes and externally binding dyes (7). The present studies suggest that dissociation of histones from DNA increases the accessibility of the anti-Z-DNA antibodies. Progression of lymphocytes through S and G6 plus M phases was associated with a decrease in binding of anti-ZDNA antibodies per unit of DNA. Enrichment of the G2 plus M population in metaphase cells did not significantly change the staining characteristics of this population, suggesting that G6 and M cells bind anti-Z-DNA antibodies to a similar extent. Furthermore, pretreatment of the cells at low pH, which increased binding of the anti-Z-DNA antibodies, did not change the relative fluorescence intensities of the G1Q-G1 versus S versus G6 plus M cells. Our data thus suggest that a decrease in the proportion of DNA with potential for Z conformation may occur when cells progress from G1 to G, plus M; this can be detected regardless of whether cells were pretreated with acid. Stimulation of G1Q cells by PHA results in increased RNA synthesis and its accumulation preceding and accompanying cell entrance into the cell cycle (8). These dramatic changes in cell metabolism and chromatin structure (4) were not paralleled by significant changes in binding of anti-Z-DNA antibodies. Surprisingly, there is high intercellular variability of the quiescent G1Q lymphocytes with respect to binding of antiZ-DNA antibodies. In contrast, quiescent lymphocytes are exceptionally uniform with respect to numerous other metabolic parameters, such as dry weight (5), RNA synthesis rate, RNA content (8), and mitochondrial content (6). Peripheral blood lymphocytes are heterogeneous with respect to cell age. T cells, which make up 70 to 80% of peripheral blood lymphocytes, are long lived, while B cells, which make up 10 to 25% of these cells, are short lived. One might speculate that Z-DNA may have a relationship to the cellular aging process. Studies on purified populations are under way to investigate this point. ACKNOWLEDGMENTS This work was supported in part by Public Health Service grants AM34485, AG00239, AG00541, GM32375, and CA28704 from the National Institutes of Health. LITERATURE CITED 1. Andrzejewski, C., Jr., J. Rauch, E. M. Lafer, B. 0). Stollar, and R. S. Schwartz. 1981. Antigen binding diversity and antiidiotype cross reaction among hybridoma autoantibodies to DNA. J.
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Immunol. 126:226-231. 2. Arndt-Jovin, D. J., M. Robert-Nicoud, D. A. Zarling, C. Greider, E. Weimer, and J. M. Jovin. 1983. Left-handed Z-DNA in bands of acid-fixed polytene chromosomes. Proc. Natl. Acad. Sci. USA 80:4344 4348. 3. Braylan, R. C., N. A. Benson, V. A. Nourse, and H. S. Kruth. 1982. Correlated analysis of cellular DNA, membrane antigens and light scatter of human lymphoid cells. Cytometry 2:337-343. 4. Darzynkiewicz, Z. 1983. Molecular interactions and cellular changes during the cell cycle. Pharmacol. & Ther. 21:143-188. 5. Darzynkiewicz, Z., V. Dokov, and M. Pienkowski. 1967. Dry mass of lymphocytes during stimulation by phytohemagglutinin. Nature (London) 214:1265-1266. 6. Darzynkiewicz, Z., L. Staiano-Coico, and M. R. Melamed. 1981. Increased mitochondrial uptake of rhodamine 123 during lymphocyte stimulation. Proc. Natl. Acad. Sci. USA 78:2383-2387. 7. Darzynkiewicz, Z., F. Traganos, J. Kapuscinski, L. StaianoCoico, and M. R. Melamed. 1984. Accessibility of DNA in situt to various fluorochromes: relationship to chromatin changes during erythroid differentiation of Friend erythroleukemia cells. Cytometry 5:355-363. 8. Darzynkiewicz, Z., F. Traganos, T. Sharpless, and M. R. Melamed. 1976. Lymphocyte stimulation a rapid multiparameter analysis. Proc. Natl. Acad. Sci. USA 73:2881-2885. 9. Drew, H. R., and R. E. Dickerson. 1981. Conformation and dynamics in a Z-DNA tetramer. J. Mol. Biol. 152:723-726. 10. Lafer, E. M., A. Molier, A. Nordheim, B. D. Stollar, and A. Rich. 1981. Antibodies specific for left-handed Z-DNA. Proc. Natl. Acad. Sci. USA 78:3546-3550. 11. Lafer, E. M., A. Moller, R. P. Valle, A. Nordheim, A. Rich, and B. D. Stollar. 1982. Antibody recognition of Z-DNA. Cold
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Spring Harbor Symp. Quant. Biol. 47:155-162. 12. Lancillotti, F., M. C. Lopez, C. Alonso, and B. D. Stollar. 1985. Locations of Z-DNA in polytene chromsomes. J. Cell Biol. 100:1759-1766. 13. Leng, M., B. Hartmenn, B. Malfoy, J. Pilet, J. Ramstein, and E. Sage. 1982. Interactions between nucleic acids and antibodies to Z-DNA. Cold Spring Harbor Symp. Quant. Biol. 47:163-170. 14. Pardue, M. L., A. Nordheim, E. M. Lafer, B. D. Stollar, and A. Rich. 1982. Z-DNA and the polytene chromosome. Cold Spring Harbor Symp. Quant. Biol. 47:171-176. 15. Pohl, F., and T. M. Jovin. 1972. Salt-induced co-operative conformational changes of synthetic DNA: equilibrium and kinetic studies with poly(dG-dC). J. Mol. Biol. 67:375-396. 16. Rich, A. 1982. Right-handed and left-handed DNA: conformational information in genetic material. Cold Spring Harbor Symp. Quant. Biol. 47:1-12. 17. Robert-Nicoud, M., D. J. Arndt-Jovin, D. A. Zarling, and T. M. Jovin. 1984. Immunological detection of left-handed Z-DNA in isolated polytene chromosomes. Effects of ionic strength, pH, temperature and topological stress. EMBO J. 3:721-731. 18. Stollar, B. D., and W. Rexuke. 1978. Separation of antihistone antibodies from nonimmune-histoprecipitating serum proteins predominant a-2 macroglobulin. Arch. Biochem. Biophys. 190:398-404. 19. Viegas-Pequignot, E., C. Derbin, E. Malfoy, E. Taillandier, M. Leng, and B. Dutrillaux. 1983. Z-DNA immunoreactivity in fixed metaphase chromosomes of primates. Proc. Natl. Acad. Sci. USA 80:5890-5894. 20. Wang, A. J., G. J. Quigley, F. J. Kolpak, G. vanderMarel, J. H. vanBoom, and A. Rich. 1981. Left-handed double helical DNA: variations in the backbone conformation. Science 211:171-176.
