26 S Proteasome-mediated Degradation of Topoisomerase II Cleavable Complexes

DNA topoisomerase II (TOP2) cleavable complexes represent an unusual type of DNA damage characterized by reversible TOP2-DNA cross-links and DNA double strand breaks. Many antitumor drugs and physiological stresses are known to induce TOP2 cleavable complexes leading to apoptotic cell death and genomic instability. However, the molecular mechanism(s) for repair of TOP2 cleavable complexes remains unclear. In the current studies, we show that TOP2 cleavable complexes induced by the prototypic TOP2 poison VM-26 are proteolytically degraded by the ubiquitin/26 S proteasome pathway. Surprisingly the TOP2β isozyme is preferentially degraded over TOP2α isozyme. In addition, transcription inhibitors such as 5,6-dichlorobenzimidazole riboside and camptothecin can substantially block VM-26-induced TOP2β degradation. These results are consistent with a model in which the repair of TOP2β cleavable complexes may involve transcription-dependent proteolysis of TOP2β to reveal the protein-concealed double strand breaks. DNA topoisomerase II (TOP2) cleavable complexes represent an unusual type of DNA damage characterized by reversible TOP2-DNA cross-links and DNA double strand breaks. Many antitumor drugs and physiological stresses are known to induce TOP2 cleavable complexes leading to apoptotic cell death and genomic instability. However, the molecular mechanism(s) for repair of TOP2 cleavable complexes remains unclear. In the current studies, we show that TOP2 cleavable complexes induced by the prototypic TOP2 poison VM-26 are proteolytically degraded by the ubiquitin/26 S proteasome pathway. Surprisingly the TOP2β isozyme is preferentially degraded over TOP2α isozyme. In addition, transcription inhibitors such as 5,6-dichlorobenzimidazole riboside and camptothecin can substantially block VM-26-induced TOP2β degradation. These results are consistent with a model in which the repair of TOP2β cleavable complexes may involve transcription-dependent proteolysis of TOP2β to reveal the protein-concealed double strand breaks. topoisomerase human TOP 4′-demethylepipodophyllotoxin thenylidene-β-d-glucoside demethylepipodophyllotoxin ethylidene-β-d-glucoside Z-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-fluoromethyl ketone 5,6-dichlorobenzimidazole riboside, CPT, camptothecin glutathione S-transferase ubiquitin-activating enzyme DNA topoisomerases are double-edged swords. They are essential for many important processes of DNA such as DNA replication, RNA transcription, chromosome condensation/decondensation, and chromosome segregation (1Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2086) Google Scholar). However, due to their delicate act on DNA, they are also highly vulnerable to xenobiotics and physiological stresses to produce topoisomerase-mediated DNA damage, mostly in the form of topoisomerase cleavable complexes (2Liu L.F. Duann P. Lin C.T. D'Arpa P. Wu J. Ann. N. Y. Acad. Sci. 1996; 803: 44-49Crossref PubMed Scopus (69) Google Scholar, 3Kingma P.S. Osheroff N. Biochim. Biophys. Acta. 1998; 1400: 223-232Crossref PubMed Scopus (81) Google Scholar, 4Nambi P. Mattern M. Bartus J.O. Aiyar N. Crooke S.T. Biochem. J. 1989; 262: 485-489Crossref PubMed Scopus (9) Google Scholar, 5Li T.K. Chen A.Y., Yu, C. Mao Y. Wang H. Liu L.F. Genes Dev. 1999; 13: 1553-1560Crossref PubMed Scopus (149) Google Scholar). So far, five human DNA topoisomerases, topoisomerase I (TOP1),1 TOP2α, TOP2β, TOP3α, and TOP3β, have been identified and characterized, and the first three have been demonstrated to be important molecular targets for antitumor drugs (1Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2086) Google Scholar, 6Champoux J.J. Dulbecco R. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 143-146Crossref PubMed Scopus (290) Google Scholar, 7Chung T.D. Drake F.H. Tan K.B. Per S.R. Crooke S.T. Mirabelli C.K. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9431-9435Crossref PubMed Scopus (258) Google Scholar, 8Hanai R. Caron P.R. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3653-3657Crossref PubMed Scopus (124) Google Scholar, 9Ng S.W. Liu Y. Hasselblatt K.T. Mok S.C. Berkowitz R.S. Nucleic Acids Res. 1999; 27: 993-1000Crossref PubMed Scopus (44) Google Scholar, 10Tsai-Pflugfelder M. Liu L.F. Liu A.A. Tewey K.M. Whang-Peng J. Knutsen T. Huebner K. Croce C.M. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7177-7181Crossref PubMed Scopus (383) Google Scholar). Both hTOP2 isozymes have been demonstrated to be the cellular targets for many clinically useful anticancer drugs such as VP-16 (etoposide) and doxorubicin (11Liu L.F. Annu. Rev. Biochem. 1989; 58: 351-375Crossref PubMed Scopus (1920) Google Scholar, 12D'Arpa P. Liu L.F. Biochim. Biophys. Acta. 1989; 989: 163-177PubMed Google Scholar, 13Errington F. Willmore E. Tilby M.J. Li L. Li G. Li W. Baguley B.C. Austin C.A. Mol. Pharmacol. 1999; 56: 1309-1316Crossref PubMed Scopus (87) Google Scholar). In the presence of these TOP2-directed drugs (TOP2 poisons), TOP2 isozymes are trapped as their covalent reaction intermediates, the reversible TOP2 cleavable complexes in which each TOP2 subunit is covalently linked to the 5′-phosphoryl ends of the four-base staggered double strand breaks (14Sander M. Hsieh T. J. Biol. Chem. 1983; 258: 8421-8428Abstract Full Text PDF PubMed Google Scholar, 15Zechiedrich E.L. Christiansen K. Andersen A.H. Westergaard O. Osheroff N. Biochemistry. 1989; 28: 6229-6236Crossref PubMed Scopus (122) Google Scholar). While the double strand breaks within the TOP2 cleavable complexes are normally concealed by TOP2, many of the cellular effects of TOP2 cleavable complexes are clearly indicative of DNA damage. For example, TOP2 cleavable complexes induced by TOP2 poisons are known to induce DNA damage responses (e.g. G2 arrest, elevation of sister-chromatid exchanges, NFκB activation, and p53 stabilization) (16Smith P.J. Soues S. Gottlieb T. Falk S.J. Watson J.V. Osborne R.J. Bleehen N.M. Br. J. Cancer. 1994; 70: 914-921Crossref PubMed Scopus (62) Google Scholar, 17Pommier Y. Kerrigan D. Covey J.M. Kao-Shan C.S. Whang-Peng J. Cancer Res. 1988; 48: 512-516PubMed Google Scholar, 18Boland M.P. Fitzgerald K.A. O'Neill L.A. J. Biol. Chem. 2000; 275: 25231-25238Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 19Moreland N. Finlay G.J. Dragunow M. Holdaway K.M. Baguley B.C. Eur. J. Cancer. 1997; 33: 1668-1676Abstract Full Text PDF PubMed Scopus (13) Google Scholar). DNA repair mutant cells (e.g. ataxia telangiectasia, progeroid Werner's syndrome, and Rad52) are also known to be hypersensitive to TOP2 poisons (20Elli R. Chessa L. Antonelli A. Petrinelli P. Ambra R. Marcucci L. Cancer Genet. Cytogenet. 1996; 87: 112-116Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 21Nitiss J.L. Liu Y.X. Harbury P. Jannatipour M. Wasserman R. Wang J.C. Cancer Res. 1992; 52: 4467-4472PubMed Google Scholar, 22Nitiss J. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7501-7505Crossref PubMed Scopus (447) Google Scholar). However, how TOP2-concealed DNA strand breaks are converted to DNA damage signals is still unknown. Inhibitor studies have suggested that both DNA replication and RNA transcription may be important for processing TOP2 cleavable complexes into DNA damage signals (23D'Arpa P. Adv. Pharmacol. 1994; 29B: 127-143Crossref PubMed Scopus (12) Google Scholar, 24D'Arpa P. Beardmore C. Liu L.F. Cancer Res. 1990; 50: 6919-6924PubMed Google Scholar, 25Bodley A.L. Huang H.C., Yu, C. Liu L.F. Mol. Cell. Biol. 1993; 13: 6190-6200Crossref PubMed Scopus (40) Google Scholar). Repair of topoisomerase cleavable complexes is conceptually challenging because of the bulkiness of the protein and the concealed nature of the breaks. However, recent studies on TOP1 cleavable complexes have suggested that both SUMO and ubiquitin pathways may be involved in repair of TOP1 cleavable complexes (26Mao Y. Sun M. Desai S.D. Liu L.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4046-4051Crossref PubMed Scopus (181) Google Scholar, 27Desai S.D. Liu L.F. Vazquez-Abad D. D'Arpa P. J. Biol. Chem. 1997; 272: 24159-24164Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). While the role of SUMO-1 conjugation to TOP1 cleavable complexes is still unclear, the role of ubiquitin conjugation to TOP1 cleavable complexes appears to trigger degradation of TOP1 via the 26 S proteasome pathway (27Desai S.D. Liu L.F. Vazquez-Abad D. D'Arpa P. J. Biol. Chem. 1997; 272: 24159-24164Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Proteolytic degradation of TOP1 cleavable complexes removes the protein bulk and presumably reveals the hidden strand breaks so that the normal DNA repair process can occur (27Desai S.D. Liu L.F. Vazquez-Abad D. D'Arpa P. J. Biol. Chem. 1997; 272: 24159-24164Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 28Desai S.D. Li T.-K. Rodriguez-Bauman A. Rubin E.H. Liu L.F. Cancer Res. 2001; 61: 5926-5932PubMed Google Scholar). Interestingly transcription inhibitors have been shown to block TOP1 degradation suggesting the involvement of RNA transcription in this particular repair process. 2S. D. Desai, D. Rodriguez-Rodriguez, and L. F. Liu, unpublished results. In the current study, we show that TOP2 cleavable complexes can also trigger ubiquitin conjugation to TOP2 resulting in 26 S proteasome-mediated degradation of TOP2. Surprisingly TOP2β is preferentially degraded over TOP2α. In addition, transcription inhibitors can substantially block TOP2β degradation. These results are consistent with a model in which repair of TOP2β cleavable complexes may involve transcription-dependent proteolysis of TOP2β to reveal protein-concealed double strand breaks. ICRF-193 was purchased from ICN Biomedicals. VM-26 was kindly provided by Bristol Myers Squibb Co. Aphidicolin, cycloheximide, and 5,6-dichlorobenzimidazole riboside (DRB) were purchased from Sigma. Z-DEVD-FMK was purchased from Calbiochem. Staphylococcal S7 nuclease was purchased from Roche Molecular Biochemicals. Antiserum against hTOP1 was obtained from scleroderma patients. Rabbit antiserum against hTOP2α was raised against the C-terminal one-third of hTOP2α (29Hwang J.L. Shyy S.H. Chen A.Y. Juan C.C. Whang-Peng J. Cancer Res. 1989; 49: 958-962PubMed Google Scholar). The anti-human TOP2β antibodies was raised by immunization with an immunogen containing GST and seven linear repeats of the peptide fragment of human TOP2β from amino acid residues 1554 to 1565 (TOP2β-(1554–1565)). The construction of the DNA fragment encoding for seven repeats of TOP2β-(1554–1565) and the synthesis of the immunogen GST-TOP2β-(1554–1565) followed the published procedure (30Hsu C.T. Ting C.Y. Ting C.J. Chen T.Y. Lin C.P. Whang-Peng J. Hwang J. Cancer Res. 2000; 60: 3701-3705PubMed Google Scholar). Briefly, the template-repeat polymerase chain reaction method was applied to the construct DNA fragment encoding multiple copies of TOP2β-(1554–1565). We designed two oligonucleotides, oligo A and oligo B. Oligo A, 5′-AAT GAA GGC GAT TAT AAC CCT GGC AGG AAA ACA TCC, encodes the target antigen (TOP2β-(1554–1565)), and oligo B, 5′-GTT ATA ATC GCC TTC ATT GGA TGT TTT CCT GCC AGG, is partly complementary to oligo A. To incorporate restriction sites for subcloning at both ends of the template-repeat polymerase chain reaction products (BamHI at the 5′-end and EcoRI at the 3′-end) as well as a stop codon at the 3′-end of the coding region, a second round of polymerase chain reaction (adapter polymerase chain reaction) with two adapter primers, primer A (5′-G ATC GGA TCC CCG GGA AAT GAA GGC GAT TAT AAC) and primer B (5′-A GCT TCT AGA ATT CTA GGA TGT TTT CCT GCC AGG) was performed. The DNA fragment encoding the seven repeats of TOP2β-(1554–1565) was subcloned into plasmid pGST-KG at the 3′-end of GST DNA. The resulting plasmid, pGST-TOP2β-(1554–1565), was introduced into XL-10 Gold, and the expressed fusion protein (GST-TOP2β-(1554–1565)) was purified by glutathione-Sepharose 4B affinity chromatography. The mouse mammary carcinoma cell line ts85 (temperature-sensitive for the ubiquitin-activating enzyme, E1) (31Finley D. Ciechanover A. Varshavsky A. Cell. 1984; 37: 43-55Abstract Full Text PDF PubMed Scopus (360) Google Scholar) was cultured in a humidified atmosphere of 5% CO2 at 30 °C in Dulbecco's minimum essential medium containing penicillin-streptomycin and 10% fetal bovine serum. FM3A, the parental cell line of ts85, was cultured under identical conditions at 37 °C. Cells were shifted to the restrictive temperature by transferring the culture dishes to a 42 °C incubator for 15 min and then maintaining at 39 °C. HeLa, human breast cancer cell ZR75-1, human lung fibroblast WI-38 and its transformed subline 2RA, and leukemic CEM and U937 cells were cultured under similar conditions at 37 °C. Cells in subconfluency were treated with 100 µm VM-26. At different times, cells were lysed by an alkali solution as (27Desai S.D. Liu L.F. Vazquez-Abad D. D'Arpa P. J. Biol. Chem. 1997; 272: 24159-24164Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Briefly, of alkali was to each and cells were by a The was by the of of and the was to The were with of a S7 nuclease reaction A, and of S7 The was at temperature for 15 nuclease 30 of a was to the The were then by and with and VM-26 a prototypic TOP2 poison that both TOP2α and TOP2β into cleavable complexes 1992; Google Scholar). studies have demonstrated that VM-26 SUMO-1 conjugation to hTOP2 Y. Desai S.D. Liu L.F. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). In the current study, we show that with VM-26 in cells in a of the In the presence of the was in A in the was with of VM-26 to The in the of was due to the presence of VM-26 on the of the Surprisingly the of hTOP2α over the for the of the be that was trapped into cleavable complexes hTOP2α by VM-26. To this a (27Desai S.D. Liu L.F. Vazquez-Abad D. D'Arpa P. J. Biol. Chem. 1997; 272: 24159-24164Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) was to TOP2 cleavable complexes in cells treated with VM-26 for min was to the of degradation of TOP2 over shown in both hTOP2α and are trapped by VM-26 into covalent complexes with as by their in an in the of nuclease of the with nuclease S7 in the of the TOP2 due to the of the covalent complexes from DNA into TOP2. These results that VM-26 the of both TOP2α and TOP2β cleavable complexes with However, TOP2β cleavable complexes are proteolytically degraded over To that the VM-26-induced of the TOP2β is a in we have many cells the human lung fibroblast cell WI-38 and its transformed subline 2RA, leukemic cell such as U937 and and breast cancer cell VM-26 was shown to induce a of the TOP2β in these However, the TOP2α to be in these in the TOP2β in different cell U937 and CEM leukemic cells were treated with VM-26 for were by as under The in this was that both hTOP2α and B, human lung fibroblast WI-38 and its subline cells were treated with VM-26. were by with and breast cancer cells were treated with VM-26. were by with and The in these was 100 The of the induced by VM-26 be due to an in the of a in the of a of To these the degradation was in the presence of the protein synthesis shown in and in the presence of was degraded in with on the VM-26-induced of the and and suggesting that VM-26-induced is due to a of TOP2 protein a we that the hTOP2α the was by suggesting that isozyme was over in the of VM-26. the VM-26-induced in the of TOP2β is due to an of degradation of TOP2β, and this process be to as TOP2β VM-26 is a of apoptotic cell death T. K. N. T. N. A. F. F. K. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). VM-26-induced degradation of TOP2β be due to the of To this we the degradation in the presence of the Z-DEVD-FMK M.J. T. G. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). shown in of Z-DEVD-FMK on VM-26-induced the of cells with the proteasome the degradation of that the 26 S proteasome an apoptotic may be involved in the degradation of To the involvement of the S proteasome we the degradation of TOP2β induced by VM-26 in a of mouse cell The ts85 cells a enzyme for ubiquitin At the enzyme is and ubiquitin conjugation to is (31Finley D. Ciechanover A. Varshavsky A. Cell. 1984; 37: 43-55Abstract Full Text PDF PubMed Scopus (360) Google Scholar). We the degradation in both ts85 cells and their parental cells The degradation of TOP2β was substantially cells were shifted to the temperature with at both VM-26 induced degradation of TOP2β in cells ubiquitin-activating involved in VM-26-induced of the mouse cell line ts85 with mutant was treated with 100 µm VM-26 for different at both the and were by with and B, the parental cells were treated with and cell were with antibodies as in The to the 26 S proteasome and the of of the that VM-26-induced TOP2β is by a ubiquitin/26 S proteasome pathway. studies have suggested that both DNA replication and RNA transcription may be involved in the processing of TOP2 cleavable complexes into DNA damage signals (23D'Arpa P. Adv. Pharmacol. 1994; 29B: 127-143Crossref PubMed Scopus (12) Google Scholar). To DNA replication RNA transcription is involved in TOP2β we have the of inhibitors on VM-26-induced TOP2β shown in of DNA replication by VM-26-induced However, of transcription by substantially and To this we also transcription camptothecin A. Wu R.S. J. Mol. Biol. PubMed Scopus Google Scholar). shown in of the breast cancer cells with VM-26-induced of To the that the is due to a of the cleavable complexes in the presence of CPT, the was performed. shown in of cells with on the of VM-26-induced TOP2β cleavable can induce DNA damage, the of degradation by also be an of a DNA damage a of transcription To this we have the of DNA damage on TOP2β shown in of cells with on VM-26-induced of transcription VM-26-induced of both and block VM-26-induced The breast cancer cells were treated with 100 µm VM-26 in the presence of 100 µm µm for and for and Cells were lysed by the procedure as under and cell were treated with nuclease S7 to from covalent and VM-26-induced of cleavable Cells were treated with VM-26 in the presence of and the as in that the with nuclease S7 was In the of the be from the covalent by and B, the of covalent complexes can be cells were treated with 100 µm VM-26 in the presence of µm for from to were by with and VM-26 is a of TOP2 cleavable on DNA. from DNA these TOP2 cleavable complexes are highly In addition, the DNA strand breaks are and protein-concealed (14Sander M. Hsieh T. J. Biol. Chem. 1983; 258: 8421-8428Abstract Full Text PDF PubMed Google Scholar, 15Zechiedrich E.L. Christiansen K. Andersen A.H. Westergaard O. Osheroff N. Biochemistry. 1989; 28: 6229-6236Crossref PubMed Scopus (122) Google Scholar). been suggested that the of TOP2 cleavable complexes is the cellular processing of TOP2 cleavable complexes into DNA damage P. Liu L.F. Biochim. Biophys. Acta. 1989; 989: 163-177PubMed Google Scholar). DNA replication, RNA transcription, and proteolysis have been to be of TOP2 cleavable complexes into DNA damage (23D'Arpa P. Adv. Pharmacol. 1994; 29B: 127-143Crossref PubMed Scopus (12) Google Scholar, 24D'Arpa P. Beardmore C. Liu L.F. Cancer Res. 1990; 50: 6919-6924PubMed Google Scholar, 25Bodley A.L. Huang H.C., Yu, C. Liu L.F. Mol. Cell. Biol. 1993; 13: 6190-6200Crossref PubMed Scopus (40) Google Scholar). However, the that of these cellular processes is involved in processing TOP2 cleavable complexes into DNA is still current results that both proteolysis and RNA transcription may be involved in the processing of cleavable complexes into DNA damage. In addition, proteolysis via the ubiquitin/26 S proteasome pathway and transcription to be VM-26-induced of TOP2β appears to be similar to of Both processes are on the of topoisomerase cleavable complexes and involve ubiquitin/26 S proteasome (27Desai S.D. Liu L.F. Vazquez-Abad D. D'Arpa P. J. Biol. Chem. 1997; 272: 24159-24164Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). In addition, both processes to be been suggested to be by TOP1 cleavable complexes and RNA polymerase of TOP1 presumably reveals the strand breaks so that repair can we that TOP2β cleavable complexes can also with RNA polymerase complexes resulting in transcription of TOP2β cleavable complexes by ubiquitin/26 S proteasome results in of the protein-concealed double strand breaks The double strand breaks can then be by R. Rev. Genet. 2001; PubMed Scopus Google Scholar, K. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). double strand breaks can trigger apoptotic cell death Soues S. F. Cancer. 1993; Google Scholar). been that proteasome inhibitors can block M. R. R. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, K. M. K. Liu Y. Biochem. Pharmacol. 2000; 60: PubMed Scopus Google Scholar). The of VM-26-induced by proteasome inhibitors is due to of NFκB M. R. R. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, K. M. K. Liu Y. Biochem. Pharmacol. 2000; 60: PubMed Scopus Google Scholar). current model the 26 S proteasome VM-26-induced the TOP2 and the of the double strand breaks. However, VM-26-induced is to be by multiple and degradation of TOP2β cleavable complexes may represent of these The degradation of TOP2β over TOP2α in cells is degradation is due to of TOP2β cleavable complexes by VM-26. appears that TOP2β cleavable complexes be by the S proteasome pathway are TOP2α cleavable The by the ubiquitin/26 S proteasome pathway may the the two These two are by in their K.B. K.M. T.D. Mirabelli C.K. Crooke S.T. Mao J. Cancer Res. 1992; 52: Google Scholar, P. T. A.L. D. Nucleic Acids Res. 1992; PubMed Scopus Google Scholar, F.H. Bartus Mattern Crooke S.T. Mirabelli C.K. Biochemistry. 1989; 28: PubMed Scopus Google Scholar). studies have shown that TOP2α is in TOP2β is in K.A. 1997; Google Scholar, K. K. O. K. A. J. 2001; PubMed Scopus Google Scholar). 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