ANALYTICAL

BIOCHEMISTRY

204,124-130

(19%)

Resolution of DNA Topoisomerase II by TwoDimensional Polyacrylamide Gel Electrophoresis and Western Blotting William

D. Wright

and Joseph

L. Roti Roti

Washington University School of Medicine, Mallinckrodt 4511 Forest Park Boulevard, St.. Louis, Missouri 63108

Received

January

Institute

Section

of Cancer Biology,

20, 1992

Eukaryotic DNA Topoisomerase II (Topo II) has been studied using high-resolution two-dimensional polyacrylamide electrophoresis (BD-PAGE) and immunodetection of resolved proteins using specific antisera (Western blotting). Traditional methods of BD-PAGE failed to resolve Topo II and neither nonequilibrium nor equilibrium pH gradients allowed Topo II to enter the first dimension gel. Exhaustive nuclease digestion and alternate protein solubilization strategies also produced negative results. We have developed altered first dimension pH gradient profiles and employed a more aggressive protein solubilization procedure which resulted in the resolution of Topo II. The 170-kDa polypeptide focuses with an apparent isoelectric point of apQ 1992 Academic Press, Inc. proximately 6.5.

DNA topoisomerases are enzymes present in the nuclei of all eukaryotes that serve to modulate DNA topology at the level of DNA supercoiling (1). DNA topoisomerase II (Topo II)’ alters DNA superhelical density and decatenates intertwined DNA by a strand passing reaction involving a protein-linked double strand break (2). The enzyme has been shown to be involved in the normal completion of S phase (3) and is essential for the segregation of chromosomes at mitosis in yeast (4). Further evidence suggests a role for Topo II in transcrip-

’ Abbreviations used: Topo II, DNA topoisomerase II; PD-PAGE, two-dimensional polyacrylamide gel electrophoresis; NEPHGE, nonequilibrium pH gradient gel electrophoresis; IEF, isoelectric focusing; pZ, isoelectric point; DTT, dithiothreitol; NP-40, Nonidet P-40; SDS, sodium dodecyl sulfate; MEM, minimum essential medium; PDA, piperazine diacrylamide; Chaps, 3-[(3-chloroamidopropyl)dimethylammonio]-1-propanesulfonate; TEMED, N,N,N’,N’-tetramethylethylenediamine; TTBS, Tris-buffered saline. 124

of Radiology,

tion, recombination, and DNA repair (5-7). In addition to its enzymatic activity, Topo II appears to play a structural role in the nuclear matrix (8) and in mitotic chromosomes (9). Recently certain antitumor drugs have been shown to be topoisomerase II poisons (10). Several of these are thought to exert their cytotoxic effects by long-term inhibition of DNA synthesis due to trapped topoisomerase II-DNA complexes (11). Due to their role in regulating DNA conformation, DNA topoisomerases have become attractive as possible modulators of cellular responses to DNA damage, particularly damage produced by ionizing radiation-a common modality in the treatment of cancer. The hypothesis has been put forth that the supercoiled status of nuclear DNA determines the efficiency with which radiation-induced DNA strand breaks are repaired by nuclear repair complexes (7,12). Thus, the study of the molecular basis for cell-cycle-dependent topoisomerase activity has become the subject of intense research (1,13). Intracellular levels of Topo II are very low in nonproliferating cells but its synthesis is stimulated when cells enter the cell cycle (14). The levels of Topo II then increase monotonically as cells traverse the cell cycle, reaching a maximum level in GJM (15). Reduction of Topo II levels to G, values in the subsequent generation is brought about by dilution among the daughter cells and by specific proteolytic degradation (15). Topo II exists in the cells as a phosphoprotein whose level of phosphorylation parallels its increased accumulation through the cell cycle, with the enzyme being hyperphosphorylated in GJM (16). To our knowledge all studies on the cell-cycle-specific amount and post-translational modification of Topo II have been performed using one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis 0003.2697192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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(SDS-PAGE) to resolve the protein. While this approach has been useful to date, the detection and quantitation of the enzyme has relied on the acquisition of specific antisera which are not commercially available. The subtle changes in charge due to phosphorylation have not been resolved without labeling the protein with 32P followed by quantification of the incorporated label. However, this method only measures total phosphorylation and cannot detect changes in the distribution of phosphorylated proteins. To partially eliminate these problems, we sought to employ the resolving power of high-resolution two-dimensional polyacrylamide gel electrophoresis (BD-PAGE) to identify Topo II. Once accomplished, the total amount of enzyme and the distribution of phosphorylation states could be assayed by measuring the optical density associated with the specific polypeptide associated with Topo II and isoelectric point shifts in stained gels. Staining intensities at various isoelectric points should allow the study of phosphorylation distribution, i.e., what fraction of the total protein exhibits various degrees of phosphorylation. MATERIALS

AND

METHODS

Cells. All experiments were performed with asynchronously growing HeLa S3 cells. The cells were grown in stirred suspension in Joklik-modified S-MEM supplemented with 3.5% each heat-inactivated fetal bovine and calf sera (GIBCO, Grand Island, NY). Cells were kept in exponential phase by daily dilution with fresh growth medium. Reagents. All reagents were of electrophoresis grade and were prepared in ultrapure type I water obtained from a Milli-Q plus water system (Millipore Corp., Bedford, MA). The following chemicals were obtained as follows: acrylamide from National Diagnostics (Highland Park, NJ); ammonium persulfate, Bis (N,N’-methylene bis-acrylamide), and PDA (piperazine diacrylamide) from Bio-Rad (Richmond, CA); ampholytes pH 3.5-10, 5-7, 6-8, 7-9, 8-9.5 from Pharmacia LKB (Piscataway, NJ); Chaps (3-[(3-cholamidopropyl)dimethylammoniol-1-propanesulfonate) from Calbiothem (La Jolla, CA); NP-40 (Nonidet P-40), SDS (specially pure) from BDH Chemicals (Poole, England); TEMED (N,N,N’,N’-tetramethylethylenediamine) from Mallinckrodt Chemical (St. Louis, MO); urea (ultra pure) from Schwarz/Mann (Orangeburg, NY). Enzymes. Deoxyribonuclease I grade DPFF, micrococcal nuclease, and ribonuclease A grade RAF, were all purchased from Worthington Biochemicals (Freehold, NJ). Isotopic labeling. Cells to be labeled with [35S]methionine were harvested from exponentially growing cultures and collected by centrifugation for 5 min at 15Og. The cells were then resuspended in methionine-free

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Joklik-modified S-MEM supplemented with 3.5% each heat-inactivated dialyzed fetal bovine and calf sera (GIBCO). [35S]Methionine (trans-35S label; Amersham, Arlington Heights, IL) was added to a final specific activity of 100 pCi/ml and the culture was incubated with gentle rocking for 4 h at 37°C. The cells were then processed for BD-PAGE. Sample preparation. For each electrophoretic condition to be studied 4-5 X lo6 cells (labeled or unlabeled) were collected by centrifugation at 150g and washed 2-3 times with Eagle’s spinner salt solution. These and subsequent steps were performed in 4°C ice baths. Washed cells were either processed for the first dimension directly or used for nuclear isolation as described (17). Cell and nuclear samples were then solubilized by a slight modification of the method described by Garrels (18). Briefly, 4-5 X lo6 cells or nuclei were collected by centrifugation for 5 min at 9OOg. After discarding the supernatant and drying the tube walls, the pellet was resuspended in 90 ~1 of an m-nuclease solution (50 pgl ml m-nuclease, 20 mM Tris, pH 8.0, 2 mM CaCl,, 0.1% NP-40) and transferred to a 1.5-ml Eppendorf microfuge tube. Immediately 10.8 ~1 of 3% SDS, 10% fl-mercaptoethanol was added followed by 10.8 ~1 of 1 mg/ml DNase I, 500 Kg/ml RNase A in 0.5 M Tris, pH 7.0, 50 mM MgCl,. Within 1 min of digestion on ice the samples were quick frozen in dry ice-methanol and lyophilized for 2-3 h. Lyophilized samples were then resuspended in 150 ~1 of sample buffer for NEPHGE (9.5 M urea, 2% NP-40,2% pH 3.5-10 ampholytes, 100 mM DTT) or IEF (9.95 M urea, 4% NP-40 & 40 mg/ml Chaps as indicated, 2% ampholytes pH 5-7, 100 mM DTT) analysis. Samples were run immediately or stored at -80°C until use. 2D-PAGE. The formulation for NEPHGE gels was 9.2 M urea, 4% acrylamide (37.5/l, acrylamide/bis-acrylamide), 2% NP-40, 2% ampholytes pH 3.5-10, and 0.5% ampholytes pH 5-7 as described by Laszlo et al. (19). IEF gels contained 9.2 M urea, 4% acrylamide (37.5/l, acrylamide/PDA), 4% NP-40 or 0.5% NP-40,15 mg/ml Chaps as indicated and 2% ampholytes of varying composition (see text). Monomer solutions were filtered through 0.2-Frn syringe filters (Gelman Sciences, Inc., Ann Arbor, MI) and degassed, and polymerization was initiated with ammonium persulfate and TEMED. First dimension tube gels (2.4 mm X 16 cm) were filled to a height of 13 cm and allowed to polymerize for 1 h. Gel surfaces were conditioned by overlaying with either NEPHGE or IEF sample buffer for 1 h. For NEPHGE, the samples were loaded immediately after conditioning and run on reverse polarity (cathode in upper chamber) in a Bio-Rad protean II electrophoresis cell at 400 V for 5 h (2000 V-h). The anolyte contained 0.01 M phosphoric acid; the catholyte contained 0.02 M NaOH. For IEF, the gels were prerun for 1 h at 200 V prior to sample loading. After loading, the gels were run at 800 V for 16 h

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FIG. 1. Localization of Topo II in 2D gels using NEPHGE in the first dimension. A, a fiuorograph of [~5S~methionine-labeied HeLa nuclear proteins separated by NEPHGE and SDS-PAGE (7.5% acrylamide). Molecular weight markers in kDa are indicated on the left. The measured pH gradient is indicated at the bottom of the gel. The area encompassed by the broken line is magnified in B. C represents a Western blot of the region shown in B using anti-Top0 II. The arrows mark the position of Topo II.

followed by 1000 V for 1 h (13,800 V-h). The anolyte and catholyte were the same composition as for NEPHGE but the gels were run on normal polarity (cathode in bottom chamber). A blank gel was run in parallel for the determination of the pH gradient (see below). After completion of the first dimension run, the gels were extruded into screw cap tubes containing 3 ml of equilibration buffer (60 mM Tris, pH 6.8,2% SDS, 100 mM DTT) and placed on a rocking platform for 2-5 min prior to loading onto the second dimension gel. The second dimension SDS slab gels were formulated according to the method of Laemmli (20). The I&cm resolving gels consisted of 7.5 or 10% acrylamide/PDA (37.5/l) as indicated. The 2-cm stacking gels consisted of 4% acrylamide/PDA. The first dimension gels were fixed to the top of the stacking gel with 1% agarose in running buffer containing 0.01% bromphenol blue as a tracking dye. The second ~mension gels were run in parallel at 50 mA/gel in a Bio-Rad multi-cell eleetrophoresis unit until the tracking dye reached the bottom of the gels. Temperature was maintained at 20°C with a constant temperature refrigerated circulating water bath connected to the multi-cell cooling coils. Protein uisualization. Gels containing radioactive proteins were impregnated with En3Hance (New England Nuclear, Boston, MA), dried onto filter paper, and exposed to Kodak X-omat AR X-ray film for 4-6 days. Nonradioactive proteins were visualized by silver staining using a silver stain kit (Accurate Chemical and Scientific Corp., Westbury, NY) according to the manufacturer’s instructions. Determination of pH Gradients. Duplicate NEPHGE or IEF gels run in parallel with protein samples

were extruded onto a glass plate and cut into l-cm pieces with a razor blade. Each piece was then equilibrated for 1 h in 1 ml of 10 mM KC1 in 5ml capped vials and then the pH of each solution was measured with a Beckman Model 31 pH meter fitted with a Futura combination electrode (Beckman Instruments, Fullerton, CA) calibrated at two different pH values (pH 4.0 and 10.0). Western blotting. Western blotting was performed using a modification of the method of Towbin et al. (21). Following completion of the second dimension run, gels to be transferred were immersed in transfer buffer (25 mM Tris, pH 8.3, 192 mM glycine, 20% methanol) for 10 min with gentle agitation. Proteins were transferred to 0.45..pm nitrocellulose sheets (Schleicher & Schuell, Keene, NH) for 16 h at 25 V at 4°C using a Hoeffer Trans-phor apparatus (Hoeffer Scientific Instruments, San Francisco, CA). Following transfer, the membranes were incubated for 2-4 h in 0.1% Tween 20 in Tris-buffered saline (100 mM Tris, pH 7.5,0.9% NaCI), hereafter referred to as TTBS, to which nonfat dry milk was added to a concentration of 5%. After blocking, the membranes were incubated for 1.5 h with a rabbit polyclonal antibody directed against the C-terminal l/3 peptide of human recombinant DNA topoisomerase II (generous gift of Dr. Leroy Liu) diluted I:500 in the same buffer to which sodium azide was added to a concentration of 0.2%. Following incubation with the primary antibody, the membranes were washed 6 X 5 min with TTBS prior to addition of a I:500 dilution of biotinylated goat-anti rabbit secondary antibody (Vector Laboratories, Burlingame, CA). After 1.5 h of incubation the membranes were washed as before and the presence of biot.inylated antibody was detected using avi-

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IEF

I

t

4.7

5.8

f

7.0

FKG. 2.

Localization of Topo II in a 2D gel using IEF in the first dimension. The lower panel represents total HeLa cellular proteins (1.3 X 10’ cells separated by IEF and SDS-PAGE (10% acrylamide) and detected by silver staining. Molecular weight markers (in kDa) are indicated on the left. The measured pH gradient is shown at the bottom of the gel. The detergent composition of the sample buffer and IEF gel was 4% NP-40, the ampholyte composition was 1.8% pH 5-7, 0.2% pH 3.5-10. The upper panel shows a Western blot of the high-molecular-weight region of the gel. The arrows indicate the position of Topo II.

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ferred to nitrocellulose and probed with anti Topo II while the other was dried and exposed to X-ray film. The fluorographs and matching Western blots were then compared in an effort to identify Topo II in the 2D protein pattern. A typical result of such an experiment is shown in Fig. 1. Figure 1A shows a fluorograph of [35S]methionine-labeled HeLa nuclear proteins separated by NEPHGE BD-PAGE. An enlargement of the high-molecular-weight region of the gel is shown in Fig. 1B. Under these conditions, most proteins of greater than lOO-kDa molecular weight failed to resolve well and appeared as streaks in the first dimension. Figure 1C shows a Western blot of the high-molecular-weight region of a duplicate gel probed with anti-Top0 II antibody. Under these conditions it can be seen that Topo II remains at the origin of the first dimension gel, indicating that it is insoluble or has an isoelectric point (~1) of less than 6.6 or was largely lost or modified during the nuclear isolation procedure. To test the last two possibilities we separated HeLa total cellular protein by IEF in the first. dimension and detected the resulting 2D polypeptide pattern by silver staining. A typical result is shown in Fig. 2. The total 2D polypeptide pattern is shown in the bottom panel. Under these IEF pH gradient conditions (pH 4.7-7.0) many proteins were well resolved but a great deal of material remained at the origin and represent either insoluble or basic proteins or protein-nucleic acid complexes. The corresponding

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RESULTS

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s.4

DISCUSSION

Ii.2

Because the isoelectric point of Topo II was unknown, we first attempted to resolve the enzyme using NEPHGE as the first dimension. Since Topo II has been shown to be enriched in the nucleus (8), we analyzed nuclear protein extracts to simplify the 2D polypeptide pattern. In order to maximize the sensitivity of detection we chose to analyze [35S]methionine-labeled proteins. Duplicate samples of [35S]methionine-labeled nuclear proteins (5 X 105 nuclei) were separated by NEPHGE and SDS-PAGE. One gel was then trans-

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FIG. 3.

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IEF pH gradient profiles generated by differing the composition of narrow range ampholytes. The detergent composition of the IEF gels was 0.5% NP-40, 15 mg/ml Chaps. All gradients contained 0.2% broad range ampholytes (pH 3.5-10). Narrow range ampholyte compositions were: curve A, 0.9% pH 5-7,0.9% pH 6-8; curve B, 1.8% pH 6-8; curve C, 1.8% pH 7-9; curve D, 1.8% pH 8-9.5.

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C FIG. 4. Localization of Topo II in 2D gels using modified IEF pH gradients. Each panel shows the 2D-PAGE pattern of whole cell HeLa proteins visualized by silver staining. The corresponding Western blot of Topo II is shown above each gel. The position of Topo II is indicated by the arrows. Molecular weight markers (in kDa) are shown on the left. The measured pH gradients are shown beneath each gel. The gels in A, B, and C correspond to the IEF pH gradient conditions shown in Fig. 3, curves B, C, and D, respectively. Sample buffer and IEF gel detergent composition was 0.5% NP-40, 15 mg/ml Chaps.

Western blot shows Topo II to be trapped within this region at the origin of the IEF gel. Since both NEPHGE and IEF 2D-PAGE failed to resolve Topo II under the conditions employedin our laboratory, we attempted to manipulate the first dimension pH gradient in an attempt to coax Topo II into a resolvable region of the gel. Also, to address the possible problem of solubility, we devised strategies to alter the detergent composition of the sample buffer and the altered pH gradient first dimension gels. Hochstrasser et al. (22) have reported achieving increased resolution of proteins (1.5 to 3 times more protein species) together with their increased solubility in their high resolution system by including the zwitterionic detergent Chaps in the sample buffer and the first dimension gels. Therefore we included this detergent in our formulations (see Materials and Methods). Alteration of the first dimension pH gradient was accomplished by varying the composition of the narrow range amphol~es. Examples of successively more basic pH gradients obtained in this fashion are shown in Fig. 3. All gradients contained 0.2% broad range (pH 3.5-10) ampholytes. The remaining 1.8% was made up of 0.9% each pH 5.7 and 6-8 ampholytes (curve A), 1.8% pH 6-8 ampholytes (curve B), 1.8% pH 7-9% ampholytes (curve C), and 1.8% pH 8-9.5 ampholytes (curve D). The gradients ob-

tained in this manner are roughly linear over a range of approximately 2 pH units. When whole HeLa cell lysates were separated by 2D-PAGE using these pH gradients in the first dimension and visualized by silver staining the results in Fig. 4 were obtained. The inclusion of Chaps in the sample buffer and first dimension formulations greatly increased sample solubility as evidenced by a reduction in protein streaking (compare Fig. 2 and Fig. 4A). Also, raising the boundary of the IEF gradient to 7.3 permitted Topo II to focus in the first dimension gel (Fig. 4A). As the pH gradients became more basic, Topo II focused further into the first dimension gel with a consistent; pl of approximately 6.5. The gels in Figs. 4A, 4B, and 4C correspond to Fig. 3 pH gradients B, C, and D, respectively. In the most extreme case (Fig. 4C), resolution was lost due to extensive protein streaking. We believe this to be an artifact of the gradient and not of protein solubility, since the same sample was used for all three conditions. Regardless, under these conditions, Topo II focuses too far to the acidic end to be resolved from other proteins. It is evident from the gels and corresponding Western blots shown in Fig. 4 that HeLa Topo II can be identified as a well-resolved major spot distant from neighboring polypeptides. It can be easily recognized as forming the lower right corner of a triangular constellation of three

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A FIG. 5. The resolution of acidic charge isoforms of Topo II by ZD-PAGE and Western blotting. A shows the 2D-PAGE polypeptide pattern of whole HeLa cell proteins visualized by silver staining. Molecular weight markers (in kDa1 are shown on the left. The area encompassed by the dotted lines is magnified in B; C shows a Topo II Western blot of the region marked by broken lines in B. Large arrows indicate the position of Topo II. Small upward pointing arrows indicate charge isoforms of Topo II. The detergent composition of the sample buffer and IEF gels is the same as described in the legend to Fig. 4. The pH gradient indicated at the bottom of B corresponds to Fig. 3, curve A.

abundant HeLa proteins. Changes in the levels of Topo II should now be measurable by quantitating the optical density of the stained polypeptide resolved by 2DPAGE, precluding the need for specific Topo II antisera. The fact that Topo II quantitatively enters the first dimension was verified by including the first dimension origin in the Western blots. Under these conditions, no detectable antigen remained at the IEF origin (for example see Fig. 5C). Under one set of conditions (Fig. 3, curve A), we were able to resolve charge isoforms of Topo II (Fig. 5). The electrophoretic conditions used in Fig. 5 differ from those used in Fig. 2 in two ways: The basic end of the pH gradient is shallower and Chaps was included in the sample buffer and first dimension gels. Here, in addition to the most abundant form of the antigen (rightmost spot), at least two additional spots of decreasing pI are well resolved. Since Topo II exists in. uiuo as a phosphoprotein, and these pI shifts are acidic in nature, we suspect that these species represent hyperphosphorylated forms of the enzyme. Work is in progress to verify the nature of these charge isoforms and to increase the resolution in this region of the IEF pH gradient. In summary, conditions have been described for the first time under which HeLa DNA Topoisomerase II can be readily identified and quantitated using high-resolution BD-PAGE and silver staining. In addition, it may be possible to monitor the extent of post-translational modification of the enzyme. The latter finding will allow quantification of changes in the abundance of Topo II in a given state of phosphorylation. This

method should be particularly useful in the study of the role of Topo II phosphorylation in the normal and perturbed cell cycle. ACKNOWLEDGEMENTS The authors thank Dr. Leroy F. Liu (The Johns Hopkins University School of Medicine) for helpful discussions, Dr. Andrei Laszlo for critical reading of the manuscript, and Mrs. Kathy Bles for manuscript preparation. Research supported by NC1 Grant CA41102. REFERENCES 1. D’Arpa, (Strauss, Jersey.

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