New monoclonal antibodies recognizing phosphorylated proteins in mitotic cells

Acta histochem. (lena) 97, 19-31 (1995) Gustav Fischer Verlag lena' Stuttgart· New York

New monoclonal antibodies recognizing phosphorylated proteins in mitotic cells Gunter Butschak', Jens Harborth 2 , Mary Osborn 2 and Uwe Karsten' 1 Max Delbriick Centre for Molecular Medicine, Berlin, Robert-Rossle-Str. 10, D-13125 Berlin, Germany and 2Max Planck Institute for Biophysical Chemistry, Gottingen, Am Faf3berg 11, D-37077 Gottingen, Germany

Accepted 27 October 1994

Summary Three monoclonal antibodies which showed strong staining of mitotic cells by screening on the human cell line MCF-7 were isolated. The antigens detected by the DH7 and BF6 monoclonal antibodies were located predominantly in multiple extranucleolar patches in interphase cell nuclei. In mitotic cells a strong increase in the fluorescence intensity was accompanied by its redistribution into a fine speckled form. Metaphase chromosomes were unstained. Centrosomes, spindle poles or midbodies were not stained either before or after extraction of the cells with Triton X-l00 under conditions which preserve microtubular structures. In immunoblots of interphase cell extracts only very few bands reacted with DH7 whereas in mitotic cell extracts - 30 bands were stained. BF6 also showed an increase in the intensity and number of bands detected in mitotic compared to interphase cell extracts, and the pattern was clearly different from that obtained with DH7. The BF6 antigen were extracted by 0.50/0 Triton X-l00, whereas the DH7 antigen was not. Dephosphorylation of the antigens strongly reduced the binding of both antibodies as measured by immunoblotting and ELISA assays. The results suggested that BF6 and DH7 detect two different phosphorylated epitopes, each of which is shared by a different subset of proteins from mitotic cells. The third antibody, BD 12, bound to several polypeptides, including one of high molecular weight that appeared to correspond to the NuMA antigen. The epitope recognized by BD 12 was not sensitive to phosphatases.

Key words: monoclonal antibodies - mitosis - immunofluorescence - immunoblotting - phosphorylation

Introduction Monoclonal antibodies (mabs) recognizing proteins which change their location during the cell cycle are useful reagents to characterize proteins which play key roles in the complex series of events during cell cycle. Examples of nuclear proteins which were first Correspondence to: G. Butschak

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defined by an immunological approach, and which show cell cycle dependence as judged by their immunofluorescence patterns, include Ki-67 (a proliferation marker exclusively expressed in dividing cells, Mr 359 and 320 kDa) (Gerdes et al., 1983, 1991; Schluter et al., 1993), and PCNA (a eyclin and accessory protein of the DNA polymerase d, Mr 36 kDa) (Lee and Hurwitz, 1991; Mathews et al., 1984; Travali et al., 1989). A second class of such proteins is found to be associated with the nuclear matrix in the interphase, but associates with components of the mitotic apparatus during mitosis. Examples include NuMA (also referred to in the past as SPN, SP-H, centrophilin, and the 1H1 antigen, Mr 240 kDa) (Compton et al., 1992; Kallajoki et al., 1993; Lyderson and Pettijohn, 1980; Maekawa and Kuriyama, 1993) and mitotin (Mr 125 kDa) (Todorov et al., 1992; Zhelev et al., 1985). In addition, the problem of modifications which may be cell cycledependent and may be common to a variety of proteins has also been approached immunologically. Thus the mabs MPM 1 and MPM 2 (Davis et al., 1983) show strong and increased staining of mitotic cells. In immunoblots of mitotic cell extracts these mabs detect a large number of proteins with Mr values ranging from 40 to > 200 kDa. The epitopes recognized by the MPM 1 and 2 antibodies are sensitive to digestion by alkaline phosphatase. Vandre et al., (1984; 1986) have shown that MPM 1 and 2 bound to centrosomes, kinetochores and midbodies. A 210 kDa protein detected by MPM 2 in isolated mitotic spindles has been identified as the phosphorylated form of MAP-4 (Vandre et al., 1991). More recently MPM 2 has been shown to bind to the centromeres and the scaffolds of isolated chromosomes, while the 170 kDa major protein band visible in immunoblots has been identified as topoisomerase IIa, and the weaker band at 180 kDa as topoisomerase lIP (Taagepera et al., 1993). Recently, the cdc25 protein has also been found to be a MPM 2 antigen (Kuang et al., 1994). The phosphorylated epitopes of two cloned MPM 2 reactive proteins have been identified by sequencing (Westendorf et al., 1994). The CC-3 mab recognizes in mitotic cells a complex set of phosphoproteins which range from 50 - 300 kDa, and in interphase cells a 285 kDa phosphoprotein (Thibedeau and Vincent, 1991 ). The MPM-12 mab defines further antigens found predominantly in mitotic cells. In immunoblots of mitotic HeLa cell extracts, bands at 155, 88, and 68 kDa have been detected, whereas in interphase cells only the 68 kDa protein is seen. Immunoblotting of all three bands is destroyed by phosphatase treatment. The 88 kDa reactive MPM-12 antigen copurifies with histone H1 kinase activity (Ganju et al., 1992). The monoclonal antibodies described above have given considerable information how proteins redistribute in interphase and mitosis. Thus the isolation and characterization of novel mabs of this type appears to be justified. Here we report results with three new antibodies: DH7, BF6 and BD12.

Materials and Methods Generation oj monoclonal antibodies. The three hybridomas were isolated during screening of fusions designed to generate antibodies to the tumour associated Thomsen-Friedenreich (TF) carbohydrate antigen. Hybridoma OH7 was derived from a Balble mouse immunized intraperitoneally (i.p.) with a sequence of alternating, TF carrying structures (MCF-7 cells, synthetic TFa and TFp coupled to BSA, asialo GMt containing Iiposomes, and human asialo erythrocytes). For the fusions resulting in OH7 and BF6 the first injection was given together with Complete Freunds Adjuvant. Hybridoma BF6 was derived from a Balb/c mouse injected Lp. with TF-BSA four times at intervals of at least one month. Hybridoma B012 was obtained from a NZB mouse immunized Lp. without adjuvant four times with living cells of the colonic carcinoma cell line Ls174T which is positive for TF antigen. In this case, spleen cells were kept frozen in liquid nitrogen before performing the fusion. The fusion partner was X63-Ag8.653 in all three cases. Cells were cultured in RPMI 1640 medium supplemented with 10 or 15010 fetal calf serum and 5x10- l M 2-mercaptoethanoL Fusions were induced with either electric field pulses (Karsten et aL, 1993) for DH7 or polyethylene glycol for BF6 and B012. Hybridomas were selected with azaserine-hypoxanthine selection

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medium. Clones were screened by immunofluorescence on MCF-7, a human breast cancer cell line. They were cloned twice by limiting dilution with microscopic control of the emerging clones. Immunoglobulin isotypes were determined with a commercial test kit (Pharmingen, San Diego/Ca, USA). Cell culture, lysate preparation and extraction conditions. Cell lines used in this study were HeLa SS6 and S3 (human cervix uteri carcinoma), MCF-7 (human mammary carcinoma), U 333CO/343MO glioma (human glioblastoma) and RMCD (rat mammary carcinoma). The cells were grown in Dulbeccos Modified Eagle's Medium supplemented with 10010 fetal calf serum and non-essential amino acids. HeLa S3 cells were grown in spinner cultures. To obtain synchronized mitotic cells, cells were grown in the presence of 2.5 mM thymidine for 18-22 h and then for a further 16 - 20 h in fresh medium containing 0.06Ilg/ml colcemid (Kallajoki et aI., 1993). The mitotic index was determined after DNA-staining by Hoechst 33258 (Hoechst AO, Frankfurt, FRO) and the fraction of dead cells was estimated after staining with Trypan blue. In the synchronized cultures the mitotic index was around 95010 whereas in the unsynchronized cultures containing > 95 010 interphase cells it was around 5010. The fraction of dead cells was -7010. The cells were centrifuged, washed three times in phosphate buffered saline (PBS) [(137 mM NaCI, 2.7 mM KCI, 4.3 mM NazHP0 4 , 1.4 mM KHzPO., pH 7.4»), and adjusted to a concentration of 4x 107 cells/ml. To minimize digestion by proteases cells harvested as above were snap frozen in liquid nitrogen, pulverized at - 70°C in a mortar, and immediately heated for 4 min at 95 °C in SDS containing sample buffer (Oerdes et aI., 1991 ). The cell lysates were diluted with SDS sample buffer to correspond to an initial cell concentration of 1x 107/ml for both mitotic and interphase cells which corresponded to protein concentrations of 1.7 and 1.2 mg/ml respectively, estimated according to Hesse et aI., (1971). The cell lysates were aliquoted and stored at - 80°C. HeLa SS6 and MCF-7 cells were washed in the culture dishes with PBS, scraped off the dishes with a rubber policeman and either pelleted and stored at - 80°C or heated for 5 min at 95 °C in SDS sample buffer. Extraction by Triton X-IOO. Harvested and washed cells were incubated at a concentration of 2 xl 07/ m! at O°C for 5 min in PBS containing 0.5010 Triton X-IOO, protease inhibitors (10 Ilg/ml aprotinin, 10 11M leupeptin, 10 11M E-64, 1 11M pepstatin, 1 mM PMSF) and 1 mM EOTA. The lysed cells were centrifuged at 13000 g for 5 min to isolate pellet and supernatant fractions which were then stored at - 80°C (Kallajoki et aI., 1993). Extraction by sonication. Cells at 2 xl 07/ ml were homogenized using a Potter-Elvehjem homogenizer and ice cold 0.1 M PIPES ( 1,4-piperazinediethanesulfonic acid) buffer, pH 6.8, containing 1 mM dithiothreitol (DTT) and the protease inhibitors as above. The homogenate was sonicated with a B 12-sonifier (Branson Sonic Power Company, Danbury, Conn., USA) using the narrow tip. Cell disruption was controlled by phase contrast microscopy. The sonicate was then centrifuged and stored as described (Kallajoki et aI., 1992). Tests on cells attached to the substrate. Immunofluorescence microscopy. Cells grown on 10 well multitest slides, or on 12 mm coverslips, were fixed in methanol at -10°C for 6 min and air dried. Cells were incubated with the primary mabs for 60 min at 37°C or at 4°C, washed three times with PBS, and incubated for 30 min at 37 DC with fluorescein-conjugated goat anti-mouse immunoglobulins (Cappel Laboratories, Cochranville, PA, USA or SIFIN, Berlin, FRO), or with rhodamine-conjugated goat antimouse 19Os (Dianova, Hamburg, FRO). After washing three times with PBS, the cells were stained for DNA with Hoechst 33258 for 1 min (20 Ilg/ml in 25010 ethanol/75010 PBS) and then mounted directly in Mowiol 4.88 (Hoechst, Frankfurt, FRO). Periodate oxidation. Carbohydrate epitopes are destroyed by periodate oxidation while peptide epitopes are not. Thus under certain conditions, this test can be used for the preliminary characterization of an unknown epitope (Woodward et aI., 1985). We applied this procedure to immunofluorescencee tests on cell lines. Cells on multitest slides were fixed with 5010 formaldehyde in PBS (5 min) and then covered with 10 mM NalO. in 0.1 M acetate buffer, pH 4.5 for I h, washed with PBS, treated with 50 mM NaBH. in PBS for 30 min, and again washed. Control cells were covered with acetate buffer without NalO•. Extraction experiments (I) Cells on multitest slides or coverslips were treated for 2 -4 min by 0.15010 Triton X-I00 in PHEM buffer (Schliwa and van Blerkom, 1981) containing 51lg/m1 taxol (Sigma, Deisenhofen, FRO). In this buffer microtubules were stabilized against the detergent treatment. After extraction the cells were washed and kept in PHEM buffer without taxol and without 1fiton X 100 for 30 min at room temperature. Then the cells were fixed in methanol and processed as described. (2) Cells were fixed in periodate-lysine-paraformaldehyde for 12 min at room temperature and then extracted with 0.1010 Triton X-I00 in PBS for 2 min at room temperature (Mclean and Nakane, 1974).

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SDS Electrophoresis and immunoblotting. SDS-PAGE was performed on 0.5 mm thick slab gels containing 7.5070 or 10070 acrylamide (Laemmli, 1970). After electrophoresis the proteins were transferred to nitrocellulose membranes (Schleicher & Schull, Dassel, FRG) after Towbin et aI., (1979) using 10070 methanol in the transfer buffer. The nitrocellulose sheets were stained reversibly with 0.5070 Ponceau S in 1070 acetic acid (Salinovich and Montelaro, 1986). After blocking (2 h at room temperature in PBS containing 4070 BSA and 0.1070 Tween 20), the strips were incubated with the primary mabs (4 °C, overnight) and then with the peroxidase conjugated secondary antibody (Dako P260, Glostrup, Denmark). The peroxidase reaction was revealed with diaminobenzidine (2 mg in 4 ml PBS) in the presence of H20 2 and 5 mM NiCh. The High Molecular Weight (HMW) Calibration Kit (Pharmacia Biotech Europe, Freiburg, FRG) and the MW-SDS-200 Kit (Sigma) were used as molecular weight standards. In Figs. 4 and 5 the standards were: 330 kDa: hog thyroglobulin subunit (Pharmacia), 205 kDa: rabbit muscle myosin subunit (Sigma), 116 kDa: E. coli-galactosidase subunit (Sigma), 67 kDa: bovine serum albumin (Pharmacia, Sigma), and 45 kDa: ovalbumin (Sigma). Digestion by phosphatases. 1. 11'eatment of nitrocellulose strips. After blotting the strips were incubated for 4 h at 37 °C with either alkaline phosphatase (EC 3.1.3.1.) from E. coli (Sigma, type III-L), calf intestine phosphatase (molecular biology quality from Boehringer, Mannheim, FRG) or acid phosphatase (BC 3.1.3.2.) from potatoes (purification degree I from Boehringer, Mannheim, FRG). Incubations with alkaline phosphatases were performed in 0.1 M 'Ifis/O.l M NaCI buffer, pH 9.0, containing 2.5 UI100 III or 48 Ull00 III of the phosphatase, respectively, and in the case of the acid phosphatase in 0.1 M Na-citrate buffer at pH 5.6 (2.5 Ull00 Ill). The buffer solutions contained protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, E64 (11lg/100 III each) and a2-macroglobulin (5 Ulloo Ill). 2. Treatment of the antigen before electrophoresis. In these experiments alkaline phosphatase from E. coli was used. The mitotic cell extract prepared by sonication (about 260 Ilg protein in a volume of 100 ml) was brought to a final concentration of 1070 SDS, vortexed, and heated for 5 min at 95 °C. After cooling, 400 ml of incubation buffer (0.1 M 'Ifis, 0.1 M NaC!, pH 9.0) and 5 Vlloo III of the phosphatase were added. After incubation for 4 h at 37 °C the volume was reduced to about 60 III in a Minicon CS15 concentrator (Amicon GmbH, Witten, FRG), and 20 III of 4 x concentrated sample buffer was added. The mixture was heated again for 5 min at 95 °C and loaded on an SDS acrylamide gel. In control experiments the extracts were incubated with phosphatase either in 150 mM phosphate buffer or in the presence of 200 mM p-glycerophosphate. Electrophoresis, blotting and immunostaining were carried out as described above. 3. ELISA experiments. Mitotic cell extract prepared by sonication was used as the antigen. It was diluted with 9 vol. of coating buffer (25 mM Na-carbonate, pH 9.6), vortexed and 50 III containing 20 Ilg protein pipetted into each well of a microtiter plate. The plates were dried overnight at 37°C. After washing three times with TBS (20 mM Tris, 150 mM NaCI, pH 7.4), incubation with phosphatase carried out for 4 h at 37°C. E. coli phosphatase was diluted to 2.5 Ull00 III and calf intestine phosphatase to 48 Ull00 III with 100 mM Tris/NaC! buffer. 50 III of enzyme solution was added to each well. Control wells contained either a) 150 mM phosphate buffer instead of the Tris buffer or b) p-glycerophosphate in a concentration of 200 mM. After phosphatase treatment the plates were washed three times with PBS, and the remaining binding sites were blocked for 30 min at 37 °C with either 4070 BSA and 0.1070 Tween 20 in PBS or with culture medium containing 10070 fetal calf serum. After incubation with the primary mabs for 2 h at 37 °C the wells were washed with PBS/O.05070 Tween 20 and incubated for 90 min at 37 °C with the second antibody (peroxidase-conjugated rabbit anti-mouse immunoglobulins, Dako P260, diluted 1 : 2000 in PBS or culture medium). The wells were washed again with PBS/1\veen and then incubated with 10 mg o-phenylene diamine and 10 III H 20 2 (30070) in 25 ml of 0.1 M citrate-phosphate buffer, pH 5.0, at room temperature. The reaction was stopped with 2.5 N sulfuric acid, and the optical density was measured at 492 nm using an ELISA reader (Spectra, SLT Labinstruments, Salzburg, Austria).

Results Hybridoma supernatants were screened using immunofluorescence microscopy and the human breast carcinoma cell line MCF-7. Three hybridomas which showed strong staining of mitotic MCF-7 cells were selected for further studies. Clones DH7, BF6 and BD 12 were subcloned twice and shown to be of the IgM, kappa isotype. Binding of the three mabs to MCF-7 cells was not affected by periodate oxidation under conditions

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which destroy carbohydrate epitopes (Woodward et ai., 1985) (data not shown). Thus the three mabs do not recognize saccharide structures, although these were the major immunogens (see above). Immunofluorescence and immunoblotting methods were used to further characterize the antigenic specificity of the three mabs. Evidence for the phosphatase sensitivity of the antigens corresponding to DH7 and BF6 were obtained both by immunoblotting and by ELISA. Immunofluorescence studies. DH7. The striking feature seen with DH7 was the very intense fluorescence associated with all stages of mitotis. This is demonstrated in Figs. 1a, c, e for MCF-7 cells in the pro-, meta-, and telophase. DNA staining of the same cells with Hoechst 33258 is shown in Figs. 1b, d, f. This antibody did not bind to metaphase chromosomes (Fig. 1c, e). The nuclei of MCF-7 and HeLa interphase cells were relatively weakly stained when compared to the brightly fluorescent mitotic cells (e.g. Figs. 1a, c) but showed a punctate stain (Fig. 1h). The same staining was also seen in nuclei of rat RMCD (Fig. 1g) and the human glioblastoma cell lines. A similar pattern was seen after extraction of HeLa or MCF-7 cells with Triton X-I00 in the presence of microtubule stabilizing buffer (Fig. 1i). Under these conditions a coarse granular staining was also apparent in the mitotic cells (compare Fig. 1j with Fig. 1e). Centrosomes, spindles or other microtubular structures were not stained. DH7 reacted not only with human and rat cells (Fig. 1) but also for example with cells of the earthworm (personal communication, M. Kasper, Technical University Dresden). BF6. The mab BF6 also stained mitotic cells very intensely as shown in Fig. 2a, c (cf. the DNA stain of the same cells in Fig. 2 b, d). Again metaphase chromosomes were not stained. Fig. 2 e shows the finer granular pattern seen within telophase cells as compared to the larger and patchy staining seen in the nuclei of interphase cells (compare for instance Fig. 2e and 1h). The staining patterns show some similarities to those obtained with DH7. However, the BF6 antigen was solubilized by Triton X-I00 extraction and appeared in this case in typically round aggregates outside the nuclei or even outside the cells. This extraction was carried out in PHEM buffer containing taxol (Fig. 2g), or after fixation in periodate-lysine-paraformaldehyde (Fig.2h). Compared to Fig.2c the granula in the mitotic cell (Fig. 2 f) appeared larger after extraction. As in the case of DH7, no staining of microtubular structures was detected with the BF6 antibody. BD12. The mab BD12 (Fig. 3a - d) bound to the poles and adjacent regions of the mitotic spindle in HeLa cells. In interphase cells, the nuclei and frequently also the centrosomes were stained (Fig. 3 b, c, d). A punctate fluorescence outlining the plasma membrane was seen especially after extraction with Triton X-I 00 (Fig. 3 b, c, d). Fig. 3e shows staining of HeLa with a NuMA specific antibody (Kallajoki et ai., 1993) and the staining of mitotic cells and interphase nuclei seems very similar to that shown for BD12 in Fig. 3 a-d. In contrast, Fig. 3 f shows the reaction with tubulin antibody on HeLa cells extracted with Triton X-1 00 in PHEM buffer. Although microtubules were well preserved under these conditions, the staining patterns of BD12 (and of DH7 and BF6) were very different from that seen with the tubulin antibody. lmmunoblotting experiments. Immunoblotting experiments of mitotic and interphase cells are shown in Fig. 4. It should be noted that both preparations contained around 5070 cells of the other type. Therefore, it may be that weak bands detectable in blots of interphase cells may stem from the 5% of mitotic cells present in the interphase preparations. DH7. A striking difference was seen in the polypeptides recognized by DH7 in interphase versus mitotic cells. In interphase HeLa S3 cells only one main band at - 255 kDa was visible (Fig. 4, lane 1). A few additional bands were detected when higher amounts of the extract were applied. By contrast in imunoblots of mitotic cell extracts around 30 bands were seen, which had Mr values ranging from - 55 kDa to > 400 kDa (Fig. 4,

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Fig. 2. Immunofluorescence studies with BF6. a, c. Different mitotic stages (meta- and anaphase) of methanol-fixed HeLa SS6 cells as compared to DNA staining of the same cells (b, d.) e. MCF-7 cells after fixation in methanol. (In this preparation the fluorescence of the anaphase cell was less intensive than in c., allowing the much lower reactivity of interphase nuclei to be demonstrated). f, g. HeLa cells after 2 min extraction with 0.15% Triton X-l 00 in PHEM buffer and fixation in methanol. h. HeLa cells after fixation in periodate-lysine-paraformaldehyde and subsequent extraction by 0.1 % Triton X-l00 (2 min). Bar = lO~m.

Fig. 1. Immunofluroescence studies with DH7. a, c, e. Different mitotic stages of MCF-7 cells (methanol fixation). b, d, f. The same cells as in a, c, e stained with the DNA-dye Hoechst 33258. g-i. Interphase cells. g. RMCD cells (fixed in methanol), h. HeLa SS6 cells (fixed in methanol and acetone), i. HeLa SS6 cells after 2min extraction with 0.15% Triton X-l00 in PHEM buffer and subsequent fixation with methanol. j. Mitotic cells (HeLa SS6, meta- and telophases) after treatment as in i. k. DNA staining of the same cells as in j. Bar = 10 Ilm.

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Fig. 3a-d. Immunofluorescence studies with BD12. a. HeLa SS6 cells after fixation in methanol. b. - d. Cells were extracted for 2min with 0.151170 lriton X-loo in PHEM buffer prior to methanol fixation. e. Immunofluorescence of HeLa SS6 cells with the NuMa antibody SPN-3. f. HeLa SS6 cells treated as in b. -d. and stained with tubulin antibody. Bar = 10 !tm.

lane 2). When a cell lysate from interphase HeLa S3 cells was treated with 0.5070 Triton X-l00 the DH7 reactive 255 kDa band remained in the pellet. If the experiments were repeated with lysates from mitotic cells, at least some of the DH7 reactive bands were found in the supernatant fraction. BF6. The number of proteins defined by BF6 was also greater in mitotic than in interphase cells (Fig. 4, lanes 3 and 4). When precautions were taken to avoid proteolysis, blots of interphase cell proteins revealed major bands at around 260 kDa and 135 kDa. Two weaker bands with Mr values of approximately 155 and 125 kDa were also seen (Fig. 4, lane 3). In blots of mitotic cell proteins these bands appeared stronger, and some 10 additional bands with molecular weights between 155 kDa and 260 kDa were detected (Fig. 4, lane 4). The immunoblots of BF6 and DH7 were clearly different. After extraction of interphase and mitotic cells with Triton X-l00 the BF6 antigens were found in the supernatant. This finding is compatible with the solubilization of the BF6 antigen seen in immunofluorescence microscopy after detergent treatment (cf. Fig. 2f - h).

Mitosis specific monoclonal antibodies 330-

330-

205-

205-

116-

116-

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6767-

45-

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Fig. 4. Comparison of interphase (lanes 1, 3, 5) and mitotic (lanes 2,4,6) HeLa 83 celllysates (7.5 mg protein/5 mm gel). Immunoblots stained with mabs DH7 (lanes 1, 2), BF6 (lanes 3, 4), and BD12 (lanes 5, 6) in the peroxidase techniques. Fig. 5. Incubation of mitotic cell extract (HeLa 83 cells, extracted by sonication, without Triton X-lOO) with alkaline phosphatase from E. coli before electrophoresis. Lanes 1, 2: DH7, lanes 3, 4: BF6, lanes 5, 6: BD12, lane 7: tubulin antibody. Lanes 1, 3, 5: Incubation with phosphatase, lanes 2, 4, 6: Incubation with phosphatase in the presence of 200 mM p-glycerophosphate.

BD12. This mab showed staining of a protein with an apparent Mr of 210 kDa which is preferentially located in mitotic cells. If a higher amount of extract was used this protein was also detected in blots of interphase cell extracts. In addition, under these conditions a 240 kDa protein weakly stained in the experiment shown in Fig. 4 (lane 6) and a 180 kDa protein appeared in both mitotic and interphase cells. Evidence for phosphatase sensivity of the antibody binding sites. Phosphatase incubation of extracts. For these studies alkaline phosphatase from E. coli was used. The incubation was performed after inactivation of possible proteases in the mitotic cell sonicate by heating to 95°C in the presence of 1% SDS. The phosphatase-treated extracts were heated in sample buffer prior to electrophoresis and immunoblotting. The results are shown in Fig. 5. Lanes 1, 2 and 3, 4 demonstrate a significant decrease in both DH7 and BF6 binding after incubation of the extracts with phosphatase. Lanes 2 and 4 show control experiments in the presence of 200 mM ,B-glycerophosphate, a strong phosphatase inhibitor. In contrast, the binding of BD12 was not influenced by treatment of the extract with the phosphatase (lanes 5, 6). Phosphatase incubation of nitrocellulose strips after blotting. These experiments were carried out with alkaline phosphatases from E. coli and calf intestine as well as with acid phosphatase from potatoes. The phosphatase treatment did not influence the binding of the three mabs on the nitrocellulose strips (data not shown), and thus we conclude that the phosphatase treatment is not effective under these conditions. ELISA experiments. The phosphatase incubation was carried out after adsorption of the mitotic cell sonicate to microtiter plates followed by probing with the mabs DH7 and BF6. The results obtained with phosphatase from E. coli (Sigma, 2.5 UII00 !ll) are shown in Fig. 6. Incubation with phosphatase abolished the binding of DH7 and led to

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DIH7

BIF6

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o ..l.-......_ - - - ' - _

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Incubation with phosphatase in 0.15 M phospbate buffer

El

Incubation with pbosphatase (fris buffer) in the presence of 0.2 M 8-glyceropbospbate

Fig. 6. Evidence for phosphatase sensitivity of the binding of mabs DH7 and BF6 to HeLa S3 mitotic cell sonicate in ELISA experiments using alkaline phosphatase from E. coli (y axis = optical density, bars = standard deviations).

a significant decrease in the binding of BF6. Incubation with alkaline phosphatase from calf intestine (Boehringer, 48 U/100 Ill) reduced the antigen binding of DH7 to about 25010 of that seen with the positive controls (data not shown). Discussion In this paper three mabs reacting with antigens which strongly stain mitotic cells are described. DH7 and BF6 recognize phosphorylated epitopes common to different subsets of proteins that become phosphorylated at mitosis. In contrast, BD12 recognizes predominantly a high molecular weight protein of Mr - 210 kDa, and the reaction is not dependent on phosphorylation. In mitotic cells a strong increase in the immunofluorescence was characteristic for both the DH7 and the BF6 antigens. In addition, both antigens were redistributed in mitotic cells. Staining of the mitotic spindle poles or centrosomes was not seen, even after extraction with Triton X-100 under conditions known to preserve microtubular structures. In interphase cells the DH7 and the BF6 antigens were located in the nuclei in the form of relatively coarse patches distinct from the nucleoli. Somewhat similar patterns have been reported for other nuclear antigens, e.g. for a 160 kDa protein after heat shock (De Graaf et aI., 1992) and perhaps also for SnRNPs (Roth et aI., 1991). In contrast to DH7, the BF6 staining was removed after extraction in situ with Triton X-I00 (cf. Figs. 1 and 2). This differential behaviour towards extraction could also be seen after centrifugation of cell extracts treated with Triton X-I00. Whereas the DH7 antigens remained in the pellet, BF6 antigens were found in the supernatant fraction. The results with DH7 and BF6 show that the two mabs recognize different protein subsets.

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Immunoblots of lysates from mitotic and interphase cells with mab DH7 confirm the conclusion drawn from the immunofluorescence studies that DH7 antigens are increased in mitotic cells. Immunoblots revealed, however, that the changes detected with this antibody during the cell cycle were complex. Thus during mitosis an array of DH7 reactive proteins appeared which were not seen in interphase cells. Since at least some of these proteins possessed a higher Mr as compared to those detected by the same mab in interphase cells, the variety of bands visible on immunoblots prepared from mitotic cells is unlikely to result from proteolysis. Significantly, most proteins which appeared in mitotic cells were no longer detectable with mab DH7 after treatment with phosphatases, suggesting that the epitope recognized by this mab is either itself phosphorylated or is strongly influenced in its conformation by phosphorylation at another site in polypeptides recognized by the DH7 mab. The results shown for DH7 suggest that the proteins which appear in mitosis and react with DH7 share a common phosphorylated epitope. In the immunofluorescence studies, BF6 and DH7 showed some similarities; however, in immunoblots there were marked differences. The BF6 antigen again showed many more immunoreactive polypeptides in mitotic than in interphase cells. The subset of proteins stained by BF6 was clearly different from that stained by DH7. Also, the two mabs showed different solubilities to detergents. A common feature of the two mabs was the sensitivity of their epitopes to phosphatase action. DH7 and BF6 can be compared to other monoclonal antibodies which recognize multiple proteins in mitotic cells, and for which the reactions are phosphatase-sensitive (see Introduction). Thus the MPM 1 and MPM 2 mabs stain the interphase nuclei of certain cell lines. MPM 2 has been shown to specifically recognize topoisomerase II ex and IIp, MAP 4, and the cdc25 protein (Kuang et al., 1994; Thagepera et al., 1993 ; Vendre et al., 1991). However, unlike DH7 and BF6, MPM 1 and MPM 2 also stain microtubulecontaining structures such as poles of the mitotic spindle, centrosomes, midbodies, and kinetochores (Vendre et al., 1984; 1986). Thus, DH7 and BF6 seem to recognize different subsets of phosphorylated proteins as do MPM 1 and MPM 2. DH7 and BF6 seem to be different from the CC-3 mab (Thibodeau and Vincent, 1991) for the same reasons. DH7 and BF6 can also be distinguished from MPM-12 since MPM-12 recognizes only 3 phosphoproteins at 155, 88 and 68 kDa in mitotic cells (Ganju et al., 1992). However, we noticed the partial similarity in immunofluorescence patterns between the DH7 and BF6 antibodies and MPM-12. The third mab BD12 shared some features with NuMA-specific antibodies. Thus in mitotic cells stained with BD12 or NuMA antibodies the poles and adjacent regions of the mitotic spindle were strongly positive in metaphase, while crescents at the poles were stained in anaphase. In interphase cells, the nuclei were stained. However, BD12 seemed to stain centrosomes in a greater proportion of cells than reported for NuMA antibodies. With NuMA antibodies, centrosomes are negative in interphase but become positive in prophase. In addition, after Triton X-100 extraction a dotted staining below the plasma membrane was visible with the BD12 antibody but has not been seen with NuMA antibodies. In immunoblots with BD12 one main band at around 210 kDa was detected in mitotic cells, with minor bands being present at 240 kDa and 180 kDa and 48 kDa if the gels were overloaded. Thus while the immunofluoresence and immunoblotting data suggest that BD12 recognizes NuMA, they suggest that the BD12 antibody may also recognize additional proteins. We also noticed that the results with BD12 were independent of phosphatase treatment of the extract. The progression from the G2-phase to mitosis is regulated by phosphorylation and dephosphorylation processes (reviewed e.g. by Solomon, 1993). However our knowledge of the phosphorylated proteins that are involved is rather limited. Mabs such as MPM 2 as well as DH7 and BF6 may help to identify some of these proteins, and thus lead to

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a better unterstanding of mitotic events. Thus future experiments will be directed at characterizing the individual phosphoproteins recognized by DH? and BF6, as well as the minor proteins recognized by BD 12. Acknowledgements

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