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Caspase-mediated Cleavage of DNA Topoisomerase I at Unconventional Sites during Apoptosis*

Open AccessPublished:February 12, 1999DOI:https://doi.org/10.1074/jbc.274.7.4335
      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.
      Eukaryotic DNA topoisomerase I (topo I),
      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 (
      • Slichenmyer W.J.
      • Rowinsky E.K.
      • Donehower R.C.
      • Kaufmann S.H.
      ,
      • Costin D.
      • Potmesil M.
      ,
      • Chen A.Y.
      • Liu L.F.
      ,
      • Gupta M.
      • Fujimori A.
      • Pommier Y.
      ,
      • Rothenberg M.L.
      ). These agents slow the resealing steps of the topo I catalytic cycle (
      • Stewart L.
      • Redinbo M.R.
      • Qiu X.
      • Hol W.G.J.
      • Champoux J.J.
      ), thereby increasing the number of covalent topo I·DNA complexes within cells (
      • Hsiang Y.H.
      • Liu L.F.
      ) and setting into motion events that result in target cell apoptosis (
      • Kaufmann S.H.
      ). In addition, topo I is an important autoantigen in rheumatic disease (
      • Shero J.H.
      • Bordwell B.
      • Rothfield N.F.
      • Earnshaw W.C.
      ,
      • Guldner H.-H.
      • Szostecki C.
      • Vosberg H.-P.
      • Lakomek H.-J.
      • Penner E.
      • Bautz F.A.
      ,
      • Maul G.
      • French B.T.
      • van Venrooij W.J.
      • Jimenez S.A.
      ). First identified as an ∼70-kDa autoantigen termed Scl-70 that reacts with sera from scleroderma patients (
      • Douvas A.S.
      • Achten M.
      • Tan E.M.
      ), topo I is recognized by sera from 25% of patients with scleroderma (
      • Bona C.
      • Rothfield N.
      ) as well as by sera from mice with the TSK (tight skin) model for this disease (
      • Bocchieri M.H.
      • Heuriksen P.D.
      • Katsuri K.N.
      • Muryoi T.
      • Bona C.A.
      • Jimenez S.A.
      ). 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 (
      • Casciola-Rosen L.
      • Rosen A.
      ).
      Apoptosis is essential for morphogenesis, tissue homeostasis, and host defense against viruses (
      • Wyllie A.H.
      • Kerr J.F.R.
      • Currie A.R.
      ,
      • Raff M.C.
      ,
      • Thompson C.B.
      , ). Although the precise biochemical pathways involved in mammalian cell death continue to receive intense scrutiny, it is now clear that cysteine-dependent aspartate-directed proteases (caspases (
      • Alnemri E.S.
      • Livingston D.J.
      • Nicholson D.W.
      • Salvesen G.
      • Thornberry N.A.
      • Wong W.W.
      • Yuan J.
      )) play important roles in the initiation and execution phases of apoptotic death (,
      • Martin S.J.
      • Green D.R.
      ,
      • Fraser A.
      • Evan G.
      ). These proteases have been shown to cleave a wide variety of cellular polypeptides located in both the cytoplasm and the nucleus (reviewed in Refs.
      • Martin S.J.
      • Green D.R.
      and
      • Miller D.K.
      ,
      • Porter A.G.
      • Ng P.
      • Jänicke R.U.
      ,
      • Cohen G.M.
      ,
      • Thornberry N.A.
      • Rosen A.
      • Nicholson D.W.
      ,
      • Villa P.
      • Kaufmann S.H.
      • Earnshaw W.C.
      ).
      Like a number of other autoantigens (
      • Casciola-Rosen L.A.
      • Anhalt G.J.
      • Rosen A.
      ,
      • Casiano C.A.
      • Martin S.J.
      • Green D.R.
      • Tan E.M.
      ), 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 (
      • Kaufmann S.H.
      ). Compared with apoptotic cleavage of other caspase targets, e.g. poly(ADP-ribose) polymerase (
      • Lazebnik Y.A.
      • Kaufmann S.H.
      • Desnoyers S.
      • Poirier G.G.
      • Earnshaw W.C.
      ) or lamin B1 (
      • Lazebnik Y.A.
      • Takahashi A.
      • Moir R.
      • Goldman R.
      • Poirier G.G.
      • Kaufmann S.H.
      • Earnshaw W.C.
      ), topo I cleavage appeared to be a later event and was typically incomplete (
      • Kaufmann S.H.
      ,
      • Casciola-Rosen L.A.
      • Anhalt G.J.
      • Rosen A.
      ). In contrast to this result, topo I fragments of 70 kDa have been reported in HeLa cells exposed to UV-B irradiation (
      • Casciola-Rosen L.A.
      • Anhalt G.J.
      • Rosen A.
      ), HL-60 cells treated with etoposide (
      • Casiano C.A.
      • Ochs R.L.
      • Tan E.M.
      ), and Jurkat cells exposed to anti-CD95 antibody (
      • Casiano C.A.
      • Martin S.J.
      • Green D.R.
      • Tan E.M.
      ,
      • Casiano C.A.
      • Ochs R.L.
      • Tan E.M.
      ). 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 (
      • Voelkel-Johnson C.
      • Entingh A.J.
      • Wold W.S.M.
      • Gooding L.R.
      • Laster S.M.
      ) and necrosis of HL-60 or Jurkat cells induced by HgCl2, ethanol, H2O2, or heat (
      • Casiano C.A.
      • Ochs R.L.
      • Tan E.M.
      ), topo I was degraded into small fragments.
      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.

      Acknowledgments

      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.

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