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Principal Research Fellow of the Wellcome Trust. To whom correspondence should be addressed: Inst. of Cell and Molecular Biology, University of Edinburgh, Michael Swann Bldg., The King's Bldgs., Mayfield Rd., Edinburgh EH9 3JR, Scotland, UK. Tel.: 44-131-650-7101; Fax: 44-131-650-7100;
* This work was supported in part by National Institutes of Health Grants AG13487 (to E. S. A.), CA69008 (to S. H. K. and W. C. E), and GM49156 (to L. S. and J. J. C.) and by a principal research fellowship from the Wellcome Trust (to W. C. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. FNf Present address: Emerald BioStructures Inc., 7865 Northeast Day Rd. W., Bainbridge Island, WA 98110. FNj Leukemia Society of America Scholar. FNk These authors contributed equally to this work.
Previous studies have demonstrated that topoisomerase I is cleaved late during apoptosis, but have not identified the proteases responsible or examined the functional consequences of this cleavage. Here, we have shown that treatment of purified topoisomerase I with caspase-3 resulted in cleavage at DDVD146↓Y and EEED170↓G, whereas treatment with caspase-6 resulted in cleavage at PEDD123↓G and EEED170↓G. After treatment of Jurkat T lymphocytic leukemia cells with anti-Fas antibody or A549 lung cancer cells with topotecan, etoposide, or paclitaxel, the topoisomerase I fragment comigrated with the product that resulted from caspase-3 cleavage at DDVD146↓Y. In contrast, two discrete topoisomerase I fragments that appeared to result from cleavage at DDVD146↓Y and EEED170↓G were observed after treatment of MDA-MB-468 breast cancer cells with paclitaxel. Topoisomerase I cleavage did not occur in apoptotic MCF-7 cells, which lack caspase-3. Cell fractionation and band depletion studies with the topoisomerase I poison topotecan revealed that the topoisomerase I fragment remains in proximity to the chromatin and retains the ability to bind to and cleave DNA. These observations indicate that topoisomerase I is a substrate of caspase-3 and possibly caspase-6, but is cleaved at sequences that differ from those ordinarily preferred by these enzymes, thereby providing a potential explanation why topoisomerase I cleavage lags behind that of classical caspase substrates such as poly(ADP-ribose) polymerase and lamin B1.
The abbreviations used are: topo I, DNA topoisomerase I; YVAD-cmk, acetyltyrosinylvalinylalanylaspartyl chloromethyl ketone; PAGE, polyacrylamide gel electrophoresis.
1The abbreviations used are: topo I, DNA topoisomerase I; YVAD-cmk, acetyltyrosinylvalinylalanylaspartyl chloromethyl ketone; PAGE, polyacrylamide gel electrophoresis.
an abundant nuclear enzyme (105-106 copies/nucleus) involved in the regulation of DNA topology and the control of gene expression, is emerging as a protein of considerable medical significance. The enzyme is an important target of camptothecin and related antineoplastic agents (
). Although the origin and significance of autoantibodies in rheumatic disease remain controversial, it has been proposed that, at least in systemic lupus erythematosus, autoantibodies may arise as a result of abnormalities in the pathway of cell death by apoptosis (
), topo I is cleaved during apoptosis. However, a contradictory picture has emerged from previous studies of topo I degradation during apoptotic execution. Initial studies indicated that topo I levels markedly diminished during etoposide-induced apoptosis of HL-60 cells without production of a discrete cleavage fragment (
). In this last model system, topo I proteolysis was inhibited by preincubation of cells with the broad spectrum caspase inhibitor benzyloxycarbonyl-VAD fluoromethyl ketone (10 μm) for 30 min before addition of anti-CD95 antibody. In contrast, during tumor necrosis factor-induced apoptosis in C3HA fibroblasts (
Several questions about the apoptotic cleavage of topo I remain unanswered. 1) Which proteases are responsible for topo I cleavage during apoptosis? 2) Where are the cleavage sites located within the topo I molecule? 3) Are the topo I fragments generated during apoptosis enzymatically active? 4) Are the same topo I fragments invariably generated in different cells undergoing apoptosis? In this study, we have mapped the sites at which caspase-3 and caspase-6 cleave topo I, compared the resulting fragments with those generated in situ in several cell types undergoing apoptosis, demonstrated that the major topo I cleavage fragment retains enzymatic activity, and probed the location of the cleaved fragment within apoptotic cells. The results of this study not only identify topo I as a caspase substrate, but also provide an explanation for its slow cleavage relative to other apoptotic events and demonstrate its variable cleavage in different apoptotic cell types.
Reagents were obtained from the following suppliers: YVAD-cmk from Bachem (Essex, United Kingdom); Hybond-C membranes, peroxidase-coupled anti-mouse and anti-human secondary antibodies, and ECL enhanced chemiluminescence reagents from Amersham International (Buckinghamshire, UK); agonistic anti-Fas (Fas is the cell-surface death receptor also known as CD95 and Apo-1) antibody CH-11 from Kamiya Biomedical Co. (Seattle, WA); etoposide, paclitaxel (Taxol®), and 5-fluoro-2′-deoxyuridine from Sigma; and polyvinylidene difluoride membrane from Bio-Rad (Hertfordshire, UK). The following antibodies were used to detect topo I by immunoblotting: C-21 murine monoclonal IgM (
); and stored at 5 mg/ml in 10 mm Tris-HCl (pH 7.5), 1 mm EDTA, 1 mm dithiothreitol, and 50% (v/v) glycerol at −20 °C. Recombinant human caspase-3 and caspase-6 were expressed in Sf9 cells as described (
). Sf9 cell extracts containing recombinant caspases were preincubated at 37 °C for 15 min with 100 μm YVAD-cmk (added from a 10 mm stock in Me2SO) or diluent. Purified topo I was then added. After a 2-h incubation at 37 °C, samples were boiled in SDS sample buffer for 5 min, subjected to SDS-PAGE (
Topoisomerase I (5 μg) cleaved as described above was subjected to SDS-PAGE on a gel containing 7.5% acrylamide and transferred to a polyvinylidene difluoride membrane. After staining with Coomassie Blue, putative cleavage products were excised and sequenced by automated Edman degradation using an ABI 476A protein sequencer.
Cell Culture and Induction of Apoptosis
Jurkat cells (kindly provided by Drs. C. M. Eischen and P. J. Leibson, Mayo Medical Center) and K562 cells (American Type Culture Collection, Rockville, MD) were cultured in RPMI 1640 medium containing 5% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin G, 100 μg/ml streptomycin, and 2 mm glutamine (medium A) at concentrations below 1 × 106 cells/ml to ensure logarithmic growth. A549 human lung cancer cells and MCF-7 breast cancer cells (from American Type Culture Collection) were passaged in medium A (A549) or in minimal essential medium containing Earle's salts, 10% (v/v) fetal bovine serum, nonessential amino acids, 1 mm sodium pyruvate, and 10 μg/ml insulin (MCF-7). MDA-MB-468 human breast cancer cells (kindly provided by Dr. Nancy Davidson, Johns Hopkins Oncology Center, Baltimore, MD) were cultured in improved minimal essential medium (Biofluids, Inc., Rockville, MD) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin G, 100 μg/ml streptomycin, and 2 mmglutamine. All cell lines were maintained in a humidified atmosphere containing 5% (v/v) CO2 and passaged at least twice weekly.
To induce apoptosis, Jurkat cells were treated with 500 ng/ml anti-Fas antibody CH-11 or 68 μm etoposide for 0–4 h as recently described (
). A549 cells treated with 68 μm etoposide or 0.2 μm topotecan for 48 h were separated into adherent (nonapoptotic) and nonadherent (apoptotic) cell populations. Likewise, adherent and nonadherent populations were isolated from MDA-MB-468 cells treated with 100 μm5-fluoro-2′-deoxyuridine (
) for 24 h, followed by a 24-h incubation in drug-free medium. All cells were washed with ice-cold serum-free RPMI 1640 medium containing 10 mm HEPES (pH 7.4) and lysed under denaturing conditions in solubilization buffer consisting of 6 m guanidine hydrochloride, 250 mm Tris-HCl (pH 8.5 at 21 °C), 10 mm EDTA, 1 mm freshly added α-phenylmethylsulfonyl fluoride, and 1% (v/v) β-mercaptoethanol as described previously (
). Aliquots containing 50 μg of protein were subjected to SDS-PAGE on gels containing 7% acrylamide, transferred to nitrocellulose, and subjected to immunoblotting. Quantitation of polypeptides on the resulting blots was performed by comparison with serial dilutions of untreated cells as described (
To induce more complete cleavage of topo I, Jurkat cells were treated with 17 μmetoposide or 100 ng/ml CH-11 antibody for 14 h. After the cells were sedimented at 200 × g for 10 min, all further steps were performed at 4 °C. Cells were washed once with RPMI 1640 medium containing 10 mm HEPES (pH 7.4) followed by phosphate-buffered saline, incubated for 20 min in nuclear isolation buffer (10 mm NaCl, 10 mm Tris-HCl (pH 7.4), and 5 mm MgSO4 containing freshly added 1 mm α-phenylmethylsulfonyl fluoride and 100 units/ml aprotinin), and homogenized in a tight-fitting Dounce homogenizer. Nuclei and nuclear fragments were sedimented at 15,000 ×g for 15 min and dissolved in solubilization buffer. The post-nuclear supernatant was sedimented at 105,000 × gfor 60 min. Protein in the supernatant from this second centrifugation step (cytosol) was precipitated with 10% trichloroacetic acid, washed once with 10% trichloroacetic acid and three times with −20 °C acetone, and dissolved in solubilization buffer.
Immunolocalization of Topoisomerase I in Apoptotic Cells
After treatment with anti-Fas antibody CH-11 or etoposide as described above, Jurkat cells were sedimented onto coverslips, air-dried, fixed in 3.7% (w/v) formaldehyde, and permeabilized with 0.2% (w/v) Nonidet P-40 (
). Topoisomerase I was visualized by indirect immunofluorescence using anti-topo I antibody C-21 and rhodamine-labeled affinity-purified anti-mouse IgM (Kirkegaard & Perry Laboratories, Gaithersburg, MD) as described previously (
). After the final series of washes, samples were incubated for 3 min with 1 μg/ml Hoechst 33258 in phosphate-buffered saline prior to mounting in Vectashield (Vector Laboratories, Inc., Burlingame, CA). Samples were visualized using a DeltaVision microscope (Applied Precision, Issaquah, WA).
RESULTS AND DISCUSSION
Cleavage of Topoisomerase I in Jurkat Cells Undergoing Apoptosis
Initial studies were performed to confirm that topo I is cleaved to a detectable fragment during apoptosis and to assess the timing of this cleavage relative to other proteolytic events. After addition of the agonistic anti-Fas antibody CH-11 to Jurkat cells, proteolytic cleavage of procaspase-7 (Fig.1D) and procaspase-3 (data not shown) was detected within 60 min, the same time frame in which we have previously demonstrated caspase activation using catalytic assays for DEVD-aminotrifluoromethylcoumarin cleavage and affinity labeling (
) to yield a characteristic 89-kDa fragment likewise was detected at 60 min (Fig.1B). Cleavage of lamin B1 (manifested in Fig.1C as a decrease in the intensity of the 67-kDa full-length polypeptide) began between 60 and 80 min after addition of CH-11 antibody to the cells. By 180 min, half of the poly(ADP-ribose) polymerase and 75% of the lamin B1 molecules were cleaved in these cells. In contrast, the earliest cleavage of topo I to a detectable fragment was not evident until 140 min after addition of CH-11 antibody to the cells (Fig. 1A). Moreover, <5% of the topo I was cleaved under these conditions. A similar disparity between cleavage of topo I and the other two caspase substrates was observed after treatment of the Jurkat cells with etoposide (data not shown). These results not only confirmed that topo I was cleaved to a detectable fragment during apoptosis initiated by triggering two discrete pathways in Jurkat cells (
). Accordingly, there were two formal possibilities that could explain the delayed cleavage of topo I. First, topo I might be cleaved by a non-caspase protease that was activated downstream of the caspases. Several groups have presented indirect evidence for such proteases (
). Second, it was possible that the delay reflected a slow rate of cleavage by one or more caspases acting at a catalytically less favorable site. This second hypothesis was consistent with the results of the experiments described below.
Although 12 human caspases have been identified to date, caspase-3 and caspase-6 are the most widely studied with respect to the cleavage of target proteins during apoptosis. To examine the possibility that caspase-3 and/or caspase-6 can cleave topoisomerase I in vitro, purified recombinant human topo I was incubated with extracts prepared from Sf9 cells infected with baculoviruses expressing either human caspase-3 or caspase-6. Each of these caspases cleaved topoisomerase I at two sites (Fig.2A). Caspase-3 generated an 80-kDa major fragment and a 76-kDa minor fragment. In contrast, caspase-6 predominantly generated the 76-kDa fragment plus a minor fragment of 82 kDa. Interestingly, neither cleavage reaction was particularly efficient, with ∼30 or 50% of the topoisomerase I remaining intact after the 2-h incubation with caspase-3 or caspase-6, respectively. This is to be contrasted with the quantitative cleavage of poly(ADP-ribose) polymerase by caspase-3 within minutes under similar conditions (
). Cleavage of topo I by each caspase was completely inhibited by preincubation of extracts with the caspase inhibitor YVAD-cmk at 100 μm (data not shown), consistent with the view that the cleavages were caspase-mediated.
To confirm that the slow rate of caspase-mediated topo I cleavage is an intrinsic property of the enzyme-substrate interaction and is not due to other factors present either in vivo or in Sf9 cell extracts, the cleavage reaction was reconstituted from purified components. Purified bovine poly(ADP-ribose) polymerase and human topo I were incubated with purified recombinant human caspase-3 in the same tube for various times at 37 °C, denatured in SDS-PAGE sample buffer, and analyzed by immunoblotting (Fig. 2B). Under these conditions, caspase-3 cleaved all of the poly(ADP-ribose) polymerase within 15 min. In contrast, topo I cleavage was undetectable at 30 min, but became evident at later time points.
Collectively, the observations in Fig. 2 (A andB) establish several points. First, topo I is cleaved by both caspase-3 and caspase-6, making it one of a small number of polypeptides that are reportedly cleaved by both caspases (
). Second, caspase-mediated topo I cleavage is relatively inefficient compared with other substrates.
Mapping the Caspase Cleavage Sites in Topoisomerase I
To further explore the caspase-mediated cleavage of topo I, the amino acid sequences of the two cleaved fragments generated by caspase-3 and the two cleaved fragments generated by caspase-6 were determined (Fig.2C). The analysis was simplified by the fact that, in each case, caspase cleavage liberated a long carboxyl-terminal fragment with a free N terminus. Thus, N-terminal sequencing of each fragment was sufficient to identify the cleavage site. The four cleaved fragments yielded three distinct cleavage sites that were defined in two cases by eight amino acids of peptide sequence and in one case by seven amino acids of sequence (underlined in Fig. 2C). This analysis revealed sites of cleavage adjacent to Asp123, Asp146, and Asp170. Cleavage occurred in the sequences PEDD123↓G (caspase-6), DDVD146↓Y (caspase-3), and EEED170↓G (caspase-3 and caspase-6). Although this analysis cannot rule out the possibility that additional caspase-mediated cleavages occur in the N-terminal domain, our kinetic analysis revealed no proteolyzed species corresponding to cleavages in this domain. Therefore, if such cleavages do occur, they either must take place after the cleavages described above or must produce species that are further processed rapidly to yield those species.
Of the three sequences, only DDVD146↓Y corresponds to a canonical caspase-3 cleavage site. The other two sequences are unusual and would not be predicted based on current understanding of caspase cleavage specificities. A recent comprehensive study employing a positional scanning substrate combinatorial library (
) demonstrated that the optimal tetrapeptide recognition sequences for caspase-3 and caspase-6 were DEVD and VEHD, respectively, in remarkable agreement with the previously mapped cleavage sites in the endogenous substrates poly(ADP-ribose) polymerase (DEVD↓G) (
). The use of these disfavored sites provides a potential explanation for the slow and incomplete cleavage of topo I (Figs. 1 and 2).
The present finding that PEDD123↓G and EEED170↓G are in fact used when caspases cleave topoisomerase I not only provides evidence that factors in addition to primary sequence are important in cleavage site selection, but also suggests that studies of caspase cleavage site specificity using small peptides might need be interpreted with caution when drawing conclusions about the cleavage of native protein substrates. This is consistent with recent results showing that caspase cleavage of poly(ADP-ribose) polymerase was significantly increased following phosphatase treatment of apoptotic extracts, whereas cleavage of tetrapeptide substrates was unaffected (
All previous studies demonstrating apoptotic cleavage of human topo I were performed using leukemia cell lines. To determine whether topo I cleavage is limited to this cell type, we examined several different cell types undergoing apoptosis in response to various stimuli. Topoisomerase I cleavage products were observed not only in Jurkat (Fig.3A, lane 5) and K562 (data not shown; identical to Jurkat cells) leukemia cells, but also in A549 lung (lanes 8 and 10) and MDA-MB-468 breast (lanes 13 and 15) cancer cells undergoing apoptosis in response to previously described proapoptotic stimuli (
). Interestingly, different patterns of topo I cleavage were observed in these model systems. In the Jurkat, K562, and A549 cell lines, the single topo I cleavage product comigrated with the major fragment (b) generated by caspase-3 in vitro (cf. Fig. 3A,lanes 2, 5, 8, and10). In contrast, apoptotic MDA-MB-468 cells contained two topo I cleavage products, a larger fragment (b) that comigrated with the major caspase-3 cleavage product and a smaller fragment (c) that comigrated with the major caspase-6 cleavage product.
To determine whether these apparent differences reflect differences between the cell lines as opposed to differences in the apoptotic stimuli, three different cell lines were treated with 100 nm paclitaxel (Fig. 3B). Consistent with the results in Fig. 3A, two topo I fragments were evident in apoptotic MDA-MB-468 cells (Fig. 3B, lane 3). As was the case for etoposide and topotecan treatment, one fragment predominated in A549 cells (Fig. 3B, lane 9). When the exposure of the blot was increased in order to begin to bring up the background, a faint smear was seen just beneath the major cleavage product (Fig. 3B, lane 9′). Although this is the region of the gel in which fragment cruns, this smear did not appear upon close inspection to correspond to a discrete band. Thus, there is a clear (although slight) difference in the pattern of topo I cleavage between MDA-MB-468 and A549 cells in response to a single initiating response. An even more dramatic difference was observed when topo I processing in apoptotic MCF-7 cells was examined. Topoisomerase I cleavage was entirely absent in these cells (Fig. 3B, lane 6), which lack caspase-3 (
). To determine whether topo I likewise undergoes relocalization, we examined the distribution of intact topo I and its fragment in apoptotic cells. Topoisomerase I has four predicted nuclear localization signals: Lys59–Glu65, Lys150–Asp156, Lys174–AspD180, and Lys192–Glu198 (Fig. 2C) (
). Although cleavage at DDVD146↓Y would remove one of these potential nuclear localization signals, the resulting carboxyl-terminal fragment would be expected to retain its nuclear targeting function. In fact, a previous study has indicated that Lys192–Glu198 is apparently sufficient to direct the nuclear transport of topo I (
Several approaches were used to examine the localization of topo I and its major fragment during apoptosis. In initial experiments, normal and apoptotic Jurkat cells were examined by indirect immunofluorescence using monoclonal anti-topo I antibody according to protocols previously described (
). In these studies (data not shown), a decrease in topo I staining was observed in many of the apoptotic cells. It appeared that the low levels of remaining detectable antigen were frequently (but not invariably) excluded from condensed apoptotic bodies. We could not, however, rule out the possibility that changes in chromatin structure were masking the topo I epitope in the apoptotic bodies, nor could we ascertain whether the topo I antibody (which detects both full-length polypeptide and cleaved fragments (Figs. Figure 1, Figure 2, Figure 3)) was providing information about the topo I that remained intact in the apoptotic cells as opposed to the topo I cleavage fragment.
To circumvent these difficulties, the distributions of topo I and its cleavage product were compared by subcellular fractionation. Jurkat cells were induced to undergo apoptosis by treatment with low levels of agonistic anti-Fas antibody or etoposide for 14 h, lysed by homogenization, and subjected to differential sedimentation to produce a sedimentable fraction (containing nuclei and nuclear fragments) and cytosol. Immunoblotting revealed that both intact and fragmented topo I were recovered exclusively in the sedimentable fraction (Fig.4A, upper panel). In contrast, procaspase-2 was exclusively cytoplasmic (Fig.4A, lower panel); and the shuttling protein B23 was found predominantly in the nucleus, but with low levels detected in the cytoplasm (middle panel). Based on these results, it appears that topo I remains associated with nuclei during apoptosis.
The Major Topoisomerase I Cleavage Fragment Remains Catalytically Active
To further examine the subcellular location of the topo I fragment as well as assess its catalytic function, a band depletion assay was performed. The basis of this assay is described in detail by Kaufmann et al. (
). Under normal conditions, the catalytic intermediate that contains the active-site Tyr723covalently linked to a 3′-phosphate of the substrate DNA has a short half-life. Recent crystallographic data suggest that the presence of the plant alkaloid camptothecin perturbs the structure of this intermediate such that the free 5′-hydroxyl of the DNA might be displaced by ∼4.5 Å from the phosphate group that would be the site of attack for religation (
). As a consequence, the religation of this intermediate is slowed; and topo I·DNA covalent complexes accumulate. If cells containing these covalent topo I·DNA intermediates are lysed under denaturing conditions and subjected to SDS-PAGE, the topo I trapped in these complexes migrates as a smear with reduced mobility, resulting in a reduction in the signal for topo I atMr ∼ 100,000.
This band depletion assay is illustrated in Fig. 4B(lanes 1–5). Treatment of control Jurkat cells with increasing concentrations of the topo I poison topotecan resulted in progressive loss of the topo I signal at Mr∼ 100,000. Control experiments revealed that the signal for topo I could be restored within 2 min by exposing the cells to conditions that shift the cleavage-religation equilibrium of the enzyme in favor of free topo I (data not shown) (
). Application of the same assay to Jurkat cells induced to undergo apoptosis by treatment with anti-Fas antibody (Fig. 4B, lanes 6–10) or etoposide (data not shown) revealed that signals for topo I and the cleavage product were both attenuated as the concentration of topotecan increased. These observations indicate not only that the topo I fragment remains in the vicinity of DNA, but also that the fragment remains catalytically active in situ, a result that is consistent with the previous finding that the amino-terminal ∼200 amino acids are dispensable for topo I activity (
). In contrast, we observed topo I cleavage to one or two relatively stable fragments in cells undergoing apoptosis after treatment with a variety of stimuli, including the topo I poison topotecan (Fig. 4). The more abundant of these fragments retains the capacity to cleave DNA and to bind topotecan (Fig. 4). These observations provide evidence for an alternative pathway of topo I degradation. Moreover, the lack of topo I cleavage in MCF-7 cells, which lack caspase-3, indicates that topo I cleavage in situ is mediated by caspase-3 or a protease downstream of caspase-3, e.g. caspase-6 (
In complementary experiments, we demonstrated that topo I is a substrate of two different caspases, caspase-3 and caspase-6 (Fig.2A). Although two other polypeptides have recently been reported to be dual substrates of these two caspases (
), topo I is the first substrate for which the cleavage sites have been identified. Two of the three cleavages mediated by caspase-3 and caspase-6 occurred at sequences that differed from those reportedly preferred by these enzymes (Fig. 2C). These results have several implications. On the one hand, the demonstration of cleavage at disfavored sequences indicates that cleavage site predictions based solely on examination of tetrapeptide substrates might not identify all caspase cleavage sites in native protein substrates. On the other hand, the use of kinetically less favorable sites might also explain the relatively slow rate of topo I cleavage by recombinant caspasesin vitro (Fig. 2, A and B) and by apoptotic proteases in situ (Fig. 1). In addition, these results raise the possibility that topo I cleavage might be used as a possible indicator of the duration of caspase activation within cells. Previous studies have demonstrated that the nuclear protein poly(ADP-ribose) polymerase is cleaved relatively soon after activation of caspase-3-like proteases in cells (
). Hence poly(ADP-ribose) polymerase cleavage indicates that caspases have been activated, but does not provide an indication of how long they have been activated. In contrast, because of its slow cleavage, topo I might be useful as an indicator of how long caspases have been active in cells.
We thank D. J. McCormick and B. J. Madden of the Mayo Clinic Protein Core Laboratory for assistance with protein sequencing; Y.-C. Cheng for the kind gift of anti-topo I antibody C-21; and C. M. Eischen, P. J. Leibson, and N. E. Davidson for kind gifts of cell lines.