Immunocytochemical Detection of DNA Synthesis in Plant Cells

JPlantPhysiol. Vol. 135.pp. 15-20{1989}

Immunocytochemical Detection of DNA Synthesis in Plant Cells HONG WANG

l

, 2,

ADRIAN

J. CUTLERZ, M. SALEEMZ, and LARRY C. FOWKEh~

Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan S7N OWO and Plant Biotechnology Institute, National Research Council, Saskatoon SlN OW9, Canada * To whom correspondence should be addressed. 1

2

Received January 17, 1989 . Accepted February 27, 1989

Summary An immunocytochemical protocol has been developed to detect DNA synthesis in plant protoplasts and cells using soybean as a model system. Cells were labelled with the thymidine analog, 5-bromo-2deoxyuridine (BrdU) and fixed for antibody treatments. Formaldehyde fixation gave better results than a mixture of methanol and acetic acid. Suspension cells required an enzyme treatment to partially remove the cell wali. Fixed protoplasts and cells were extracted with Triton X-100 and reacted with a monoclonal antibody against BrdU. The BrdU labelled nuclei were visualized with either enzyme- or fluorescein-conjugated secondary antibodies. Aphidicolin reduced the number of nuclei labelled by more than 50 % at 10 J.l.g' ml- I , and inhibited the labelling completely at 50 J.l.g . ml- I , confirming that the labelling was due to replicative nuclear DNA synthesis. Immunocytochemistry is simpler than autoradiography and allows simultaneous detection of DNA and other antigens including, for example, microtubules. This technique should be widely useful in studies of plant DNA replication. Key words: Glycine max L.; aphidicolin; 5-bromo-2-deoxyundine (BrdU); DNA replication; immunofluorescence microscopy; protoplast.

Introduction Cellular DNA synthesis has been a major area of biological investigations. Studies of DNA synthesis in proliferative cells have usually involved using radioactive substrates (e_g. 3H-thymidine). The detection of the radioactivity incorporated into newly synthesized DNA relies on either autoradiography or trichloroacetic acid precipitation followed by liquid scintillation counting (Taylor et ai., 1957; Birks, 1964; Baserga and Malamud, 1969). The former is a relatively time consuming and difficult technique. The latter does not provide information on the sites of DNA synthesis and the frequency of replicating cells. In addition, precautions regarding handling radioactive materials have to be taken and radioactive wastes are inevitably produced. Immunocytochemistry offers one alternative for study of DNA synthesis by using a polyclonal or monoclonal antibody to detect 5-bromo-2-deoxyuridine (BrdU) incorporated © 1989 by Gustav Fischer Vedag, Stuttgart

into DNA (Gratzner et ai., 1975; Gratzner, 1982). It has been demonstrated that BrdU, an analog of thymidine, can be incorporated into DNA in the presence of 5-fluoro-2-deoxyuridine (FrdU), which inhibits thymidine incorporation (Ellwart and Dormer, 1985). An antibody is used to probe for incorporated BrdU and detection follows after treatment with an enzyme- or fluorescein-conjugated secondary antibody. This approach has been successfully used with animal cells for detection (Gratzner et al., 1975, 1982) and quantification of DNA synthesis (Dolbeare et ai., 1983), and also studies on sister-chromatid exchange (Morstyn et ai., 1984) and cell kinetics (Raza et ai., 1984; Campana et ai., 1988)_ The technique has a number of advantages; it is rapid, provides information on the sites of synthesis and does not involve radioactive materials. In the present communication a method for immunochemical detection of DNA synthesis in plant cells is described.

HONG WANG, ADRIAN J. CUTLER, M. SALEEM, and LARRY C. FOWKE

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Materials and Methods Cell line The soybean [Glycine max (L.) Merr.] cell line (SB-l) was established in 1966 (Gamborg). It consists of small cell clusters capable of rapid cell division. Cells were cultured in l-B5 medium (Gam borg, 1982) as described by Saleem and Cutler (1986).

Protoplast culture and incorporation ofBrdU Two days after subculture cells were resuspended in enzyme solution consisting of 0.6 % cellulase Onozuka R-l0 (Kanematsu-Gosha Ltd., CA, USA), 0.2 % Driselase (Plenum Scientific Research Inc., Hackensack, NJ, USA), 0.2 % hemicellulase (Rohm and Haas Co., Canada Ltd., West Hill, Onto Canada) and 0.2 % pectinase (Terochem Laboratories Ltd., Edmonton, Alberta, Canada) in 0.55 M sorbitol with pH adjusted to 5.8 and incubated in darkness on a shaker (approx. 50 rpm) for 12 - 14 h. The resulting protoplasts were filtered through a 48 pm nylon mesh. After washing twice in 0.55 M sorbitol solution (PH 5.8), protoplasts were resuspended at a density of 4 x 105 • ml- 1 in the protoplast culture medium (Kao, 1982) with or without 0.1 % of the labelling reagent (Amersham, see below) and cultured in a thin layer in tissue culture Petri dishes (60 x 15mm). Protoplasts were fixed at 0,1,4,13 and 26h after culture.

Detection of BrdU using peroxidase-conjugated antibody Required chemicals were obtained as a kit from Amersham International (Buckinghamshire, UK) containing a labelling reagent (with BrdU and FrdU in ratio of 10: 1), mouse monoclonal antiBrdU antibody, peroxidase-conjugated rabbit anti-mouse IgG antibody, 3,3'-diaminobenzidine tetrahydrochloride (DAB), and a substrate and intensifier reagent. The reagents were used as described by Amersham with minor modifications. Procedure I. Freshly isolated protoplasts were fixed in 3.7% formaldehyde at 24°C in a buffer (MtSB) containing 100 mM 1,4-piperazinediethylsulfonic acid (Pipes), 1 mM MgS04 and 2 mM ethylene glycol-bis-(aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), pH 6.9, for 8 h. Protoplast cultures (> 0.5 days) were treated with the above fixative containing 0.1 % Triton X-l00 for 4 h. The fixed protoplasts and cells were attached to coverslips previously coated with poly-L-Iysine (MW 564,000, Sigma Chemical Co., St. Louis, MO, USA) and extracted in 1 % Triton X-l00 in MtSB for 1 h. They were then treated with 1.5 % H 20 2 in phosphate buffered saline (PBS) (O.14M NaCI, 2.7mM KCI, 8.0mM Na2HP04, 1.5mM KH2P04, pH 7.0) for 20 min and rinsed three times in PBS each three min. A 100/LI aliquot of monoclonal mouse anti-BrdU antibody was layered on each coverslip and incubated for 60 min, followed by three rinses in PBS. The coverslip was then treated with 100 ~ peroxidase-conjugated rabbit anti-mouse IgG antibody and rinsed three times in PBS. Cells were stained with DAB in PBS for 5 min and rinsed in PBS. Controls included the cells cultured without BrdU and also cells processed without the first antibody. Variations of this procedure included: 1) omission of extraction in 1 % Triton X-l00, and 2) staining of nuclei with Hoechst 33258 (10/Lg· ml- 1 in PBS, Aldrich Chern. Co., Milw, WI, USA) after DAB staining. Procedure II. Protoplasts were fixed in a mixture of methanol and acetic acid (3: 1) (Gratzner, 1982) for 1 h. After attachment on polyIysine-coated coverslips they were treated in 1.5 % H 202 in methanol for 20 min. Staining of the labelled nuclei was the same as in procedure I.

Detection of BrdU using fluorescein-conjugated antibody Cells were processed as described above except for the following modifications: replacement of treatment in H 20 2 with treatment in 0.1 % NaBH4 in PBS for 6min to neutralize the excess aldehyde, replacement of peroxidase-conjugated antibody with fluoresceinconjugated rabbit anti-mouse IgG antibody (Amersham) diluted 1 : 50. Nuclei were couterstained with Hoechst 33258. For simultaneous staining of microtubules, a mouse monoclonal anti-chicken tubulin antibody (Sigma) was used in a dilution of 1 : 150 along with the primary antibody against BrdU and followed by the secondary antibody described above.

Cells from suspension cultures Two days after subculture an aliquot of cells was subcultured in l-B5 medium containing 0.1 % of the labelling reagent. Labelled and unlabelled cells were fixed in 3.7 % formaldehyde in MtSB with 0.1 % Triton X-l00. They were incubated in enzyme solution for 2 h and then stained as described above.

Inhibition of nuclear DNA synthesis by aphidicolin To confirm the results of nuclear DNA labelling with BrdU, aphidicolin, an inhibitor of DNA polymerase-ex (Ikegami et al., 1978; Ohashi et al., 1978; Pedrali-Noy and Spadari, 1979) was supplemented in the protoplast culture medium at 0, 1, 5, 10 and 50 /Lg. ml- 1• Aphidicolin was added from a 10 mg· ml- 1 stock in dimethyl sulfoxide (DMSO). Protoplasts were cultured for 24 h. The controls included protoplasts cultured in the medium with DMSO added.

Light microscopy Cells were examined using a Zeiss epifluorescence microscope equipped with an FITC filter set ( # 487709) for detection of the antibody staining and a filter set (#487703) for Hoechst 33258 staining. Micrographs were recorded on XPl-400 film.

Results and Discussion Peroxidase-conjugated antibody After fixation with procedure I, labelled nuclei were darkly stained and only a faint non-specific staining background appeared in unlabelled nuclei and cytoplasm of some protoplasts (Figs. 1 a and 1 b). Nuclei were either unstained or stained poorly with procedure II. Out of three experiments only one resulted in some nuclear staining (Fig. 1 c), but the frequency of stained nuclei was considerably reduced from results obtained with procedure I. Methanol! acetic acid fixation also tended to produce higher non-specific background in the cytoplasm. In both procedures nuclei were not specifically stained if cells were cultured in the absence of BrdU or if the primary antibody was omitted from the staining procedure (Fig. 1 d). In procedure I, protoplasts cultured less than half a day were fixed in formaldehyde lacking Triton X-100 to preserve morphology. These protoplasts had little or no cell walls and were subsequently lysed by detergent treatment prior to fixation. Subsequent extraction (permeabilization) was necessary to stain labelled nuclei (Fig. 1 e). For cultures more than half a day old, which were fixed in the presence of 0.1 %

Immunocytochemical detection of DNA synthesis in plant cells

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Fig. 1: ImmunolOgiCal aetectlon or Ul'"ljj\ syntnesls m soyoean protoplasts usmg peroxloase-conJugateo antloooy. l'rotoplasts were culturea in a medium containing BrdU. The BrdU labelled nuclei were visualized by a color reaction catalyzed by peroxidase. All micrographs X215 except for b X850. a. Cultured protoplasts prepared for staining by procedure 1. The labelled nuclei were darkly stained and the unlabelled nuclei and cytoplasm showed very little staining. b. Cultured protoplasts as in b shown at higher magnification. c. Protoplasts prepared for staining by procedure II. There was weaker staining in the nuclei and higher non-specific staining. d. No nuclei stained when the primary antibody against BrdU was omitted from the staining procedure. e. Protoplasts fixed without Triton X-100. Nuclei were not stained without extraction in Triton X-100 after fixation. f. Higher non-specific staining when treatment in H 20 2 was omitted.

Triton X-l00, nuclei could be stained without further detergent treatment. However, extraction in 1 % Triton X-l00 was routinely performed to reduce non-specific staining. If samples were not pre-treated in H 2 0 2 , to inhibit endogenous peroxidase activity, non-specific staining resulted (Fig. 1 f). The concentrations of the primary and secondary antibodies could be reduced by a factor of 2 with no appreciable reduction in response.

Fluorescein-conjugated antibody Using procedure I, labelled nuclei could also be detected by use of a fluorescein-conjugated second antibody. This procedure resulted in clear and bright nuclei with little nonspecific fluorescence in the cytoplasm and unlabelled nuclei (Fig. 2 ai). All nuclei were stained with Hoechst 33258 (Fig. 2 aii), thus distinguishing the labelled nuclei and unlabelled nuclei. Observation was conveniently accomplished by switching back and forth between the appropriate filters on the microscope. Also some structural details were visible, as demonstrated by the shape of nucleoli or the presence of in-

dividual chromosomes when cells were dividing (Fig. 2 bi). When cultures were fixed by methanol! acetic acid, there were either no stained nuclei (Fig. 2 ci) or only faint fluorescent nuclei when antibodies were applied at the same concentrations as in procedure I. In addition, there was very strong autofluorescence with the filter system used for detecting Hoechst 33258 staining (Fig. 2 cii). Cells processed with procedure I could also be stained for microtubules (Fig. 2 di). Since both primary antibodies were raised in mouse, only one secondary antibody was needed to achieve double labelling. Fixation of materials is essential for satisfactory antigen detection (Brandtzaeg, 1982). Although the effect of different fixation procedures has not been fully explored in plant cells (Knox, 1982), fixation with aldehyde-based fixatives i.e. formaldehyde and glutaraldehyde, adequately preserves morphology. The fixation conditions described here, using formaldehyde, proved better than fixation with methanol/ acetic acid for detection of BrdU labelled nuclei. It appeared that fixation with methanol and acetic acid reduced the ability of the antigen to react with the antibody. Similar results

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HONG WANG, AoJUAN]. CUTLER, M. SALEEM, and LARRY C. FOWKE

Fig.2: Immunocytochemical detection of DNA synthesis in soybean protoplasts using fluorescein-conjugated antibody. Cultured protoplasts were fixed, extracted and reacted with the antibody against BrdU. The BrdU labelled nuclei were visualized by a fluorescein-conjugated secondary antibody and all the nuclei were counterstained with Hoechst 33258. The first one of each set of micrographs shows the antibody staining and the second, the Hoechst staining. a-c, X850; d, X600. a. Protoplasts fixed by procedure 1. Two nuclei were visualized by the fluorescent antibody treatment (i) and an unlabelled nucleus could be seen with Hoechst staining. b. A dividing cell with labelled chromosomes. c. Protoplast treated with procedure II. The nucleus was not stained with the antibody (i) and there was a considerable amount of autofluorescence when Hoechst staining was visualized (ii). d. Simultaneous detection of DNA synthesis and microtubules. One cell had a preprophase band of microtubules (arrow) and the other cell had a labelled nucleus.

were obtained with maize leaf protoplasts (unpublished data). It is not surprising that plant cells require different optimal fixation conditions from animal cells for detection of antigens. For these cells formaldehyde fixation is also superior to glutaraldehyde fixation since the latter resulted in higher non-specific fluorescence (unpublished observation).

Suspension cells . DNA synthesis could be detected using the same method in suspension cells (Fig. 3 a). However, cells required treatment in enzyme solution after fixation to partially remove the cell wall. Sometimes there was quite high non-specific staining when peroxidase-conjugated antibody was used, presumably due to endogenous peroxidase activity (Fig. 3 b). This background was eliminated by using fluorescein-conjugated antibody (Figs. 3 c-d). The high background was usually not present in cultured protoplasts, probably because the endogenous peroxidase activity was reduced more significantly by either the detergent extraction orthe H 20 2 treatment.

Time course of BrdU incorporation in soybean protoplast cultures The percentage of labelled nuclei at various times after protoplast culture is shown in Fig. 4. No nuclei were stained before adding the labelling reagent. About 9 % nuclei were labelled after 1 h, indicating efficient incorporation of BrdU during DNA synthesis. Therefore, experiments with pulse labelling should be possible using this technique in place of traditional thymidine incorporation to assess rates of DNA replication for desired periods (e.g. Okada et aI., 1982). The percentage of labelled nuclei increased up to one day, indicating that the incorporation of BrdU during continuous labelling was not noticeably reduced.

Inhibition of BrdU incorporation by aphidicolin The % of labelled nuclei, as shown in Fig. 5, decreased steadily with increasing aphidicolin concentration while controls did not show any obvious reduction, demonstrating

Immunocytochemical detection of DNA synthesis in plant cells

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Fig. 3: Detection of DNA synthesis in suspension cells. a- b. Nuclei were stained using peroxidase-conjugated antibody, X850; c-d. Nuclei were stained using fluorescein-conjugated antibody, X580. a. Two stained nuclei and one unstained nucleus. b. Stained nucleus with a high level of non-specific staining in the cytoplasm. c. Nuclei labelled with the fluorescent antibody. d. Hoechst staining of the same cells as in c.

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Fig.4: Time course of BrdU incorporation in soybean protoplasts. The freshly isolated protoplasts were cultured for 0, 1,4, 13 and 26 h in the presence of BrdU and FrdU. Cells were fixed and stained and the percentage of BrdU labelled nuclei determined. For one treatment, about 200 nuclei from each of four random samples were examined.

unambiguously that nuclear labelling was due to nuclear DNA synthesis. After 24 h, more than 55 % of nuclei were labelled in the absence of aphidicolin. This is in agreement with the data in Fig. 4. Aphidicolin at 10 p.g . ml reduced the % of labelled nuclei by slightly more than 50 %. The complete inhibition was achieved by 50 p.g . ml aphidicolin. Thus the effect of an inhibitor can be expressed as the number of nuclei where the incorporation is inhibited rather than a reduction of overall incorporation of 3H-thymidine as in liquid scintillation counting. It should be pointed out that 50 % reduction of labelled nuclei in this case is not necessarily equal to 50 % reduction of incorporation as determined by 3H-thymidine. These results also imply a variation in sensitivity of nuclei to aphidicolin inhibition.

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Fig. 5: Effect of aphidicolin on the incorporation of BrdU. The freshly isolated soybean protoplasts were cultured in the presence of BrdU at various concentrations of aphidicolin. After 24 h cells were fixed and stained for the percentage of labelled nuclei. In one treatment a sample of 200 nuclei from each of four replicate cultures were examined. The horizontal axis is presented after a log(X + 1) transformation.

The current method presents an efficient and non-radioactive detection of DNA synthesis in plant cells and protoplasts. Peroxidase-conjugated antibody can be used and thus a fluorescent microscope is not required. Alternatively use of a fluorescein-conjugated antibody eliminated non-specific staining. In addition, the method can be used for simultaneous immunochemical detection of microtubules and might be generally useful for detection of multiple antigens. In view of its efficiency, non-radioactivity, simplicity and versatility this method is likely to be widely used in studies of DNA synthesis of plant cells including quantification.

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HONG WANG, ADRIAN]. CUTLER, M. SALEEM, and LARRY C. FOWKE

Acknowledgements The authors are grateful to Ms. M. Loewen for maintaining the cell culture. H. W. is supported by a scholarship from the University of Saskatchewan. The work was partially supported by an operating grant (A 6304) from the Natural Science and Engineering Council of Canada to L. C. F.

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GRATZNER, H. G., R. C. LEIF, D.]. INGRAM, and A. CASTRO: The use of antibody specific for bromodeoxyuridine for the immunofluorescent determination of DNA replication in single cells and chromosomes. Exp. Cell Res. 95, 88-94 (1975). IKEGAMI, S., T. TAGUCHI, M. OHASHI, M. OGURO, H. NAGANO, and Y. MANO: Aphidicolin prevents mitotic cell division by interfering with the activity of DNA polymerase-a. Nature 275, 458-460 (1978). IUo, K. N.: Plant protoplast fusion and isolation of heterokaryocytes. In: Plant Tissue Culture Methods (WETTER, L. R. and F. CONSTABEL, eds.), pp. 49-56. Saskatoon, Saskatchewan: Prairie Regional Laboratory, National Research Council Canada (1982). KNOX, R. B.: Methods for locating and identifying antigens in plant tissues. In: BULLOCK, G. R. and P. PETRUSZ (eds.): Techniques in Immunocytochemistry, Vol. 1, pp. 205-238. Academic Press, London (1982). MORSTYN, G., S.-M. Hsu, T. KINSELLA, H. A. GRATZNER, and ]. B. MITCHELL: Bromodeoxyuridine in tumors and chromosomes detected with a monoclonal antibody. J. Clin. Invest. 72, 1844-1850 (1983). OHASHI, M., T. TAGUCHI, and S. IKEGAMI: Aphidicolin: A specific inhibitor of DNA polymerases in the cytosol of rat liver. Biochem. Biophys. Res. Comm. 82, 1084-1090 (1978). OKADA, K., 1. TAKEBE, and T. NAGATA: Expression and integration of genes introduced into highly synchronized plant protoplasts. Mol. Gen. Genet. 205, 398-403 (1986). PEDRALI-Noy, G. and S. SPADARI: Effect of aphidicolin on viral and human DNA polymerases. Biochem. Biophys. Res. Comm. 88, 1194-1202 (1979). RAZA, A., H. D. PREISLER, G. L. MAYERS, and R. BANKERT: Rapid enumeration of S-phase cells by means of monoclonal antibodies. N. Engl. J. Med. 310, 991 (1984). SALEEM, M. and A. ]. CUTLER: Preparation and characterization of chemically and osmotically permeabilized soybean [Glycine max (L.) Merr.] protoplasts. J. Plant Physiol. 124, 11-21 (1986). TAYLOR,]. H., P. S. WOODS, and W. L. HUGHES: The organization and duplication of chromosomes as revealed by autoradiographic studies using tritium-labelled thymidine. Proc. Nat!. Acad. Sci. USA 43, 122-127 (1957).