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Bent Ejlertsen - One of the best experts on this subject based on the ideXlab platform.
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CEF is superior to CMF for tumours with TOP2A aberrations: a Subpopulation Treatment Effect Pattern Plot (STEPP) analysis on Danish Breast Cancer Cooperative Group Study 89D.
Breast cancer research and treatment, 2010Co-Authors: Katrín Á Gunnarsdóttir, Maj-britt Jensen, David Zahrieh, Richard D. Gelber, Ann Knoop, Marco Bonetti, Henning T. Mouridsen, Bent EjlertsenAbstract:The aim of this study was to examine TOP2A gene copy number changes as a means to identify groups of breast cancer patients with superior benefit from treatment with anthracyclines. Tumour tissue was retrospectively collected and successfully analysed for TOP2A in 773 of 980 Danish patients randomly assigned to receive intravenous CMF (cyclophosphamide, methotrexate and fluorouracil) or CEF (cyclophosphamide, epirubicin and fluorouracil) in DBCG trial 89D. Subgroup analyses on this material published by Knoop et al. (J Clin Oncol 23:7483–7490, 2005) and updated by Nielsen et al. (Acta Oncol 47:725–734, 2008) demonstrated that superiority of CEF over CMF is limited to patients with TOP2A aberrations, defined as patients whose tumours have TOP2A ratio below 0.8 or above 2.0. The Subpopulation Treatment Effect Pattern Plot (STEPP) technique was applied to these data to explore the pattern of treatment effect relative to TOP2A and to compare that pattern to the ranges previously used to define ‘aberrations’. The pattern of treatment effect illustrated by the STEPP analysis confirmed that the superiority of CEF over CMF is indeed limited to patients whose tumours have high or low TOP2A ratios. The hypothesis of no treatment effect–covariate interaction was rejected (P = 0.02). Furthermore, results indicated that the interval of TOP2A ratios hitherto denoted as ‘normal’ could be narrower than previously assumed. A more optimal separation of TOP2A subgroups could be obtained by altering cut-points currently used to define TOP2A amplified and TOP2A deleted tumours by narrowing the TOP2A normal interval, and consequently enlarging the population with TOP2A aberrated tumours.
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aberrations of erbb2 and TOP2A genes in breast cancer
Molecular Oncology, 2010Co-Authors: Kirsten Vang Nielsen, Ann Knoop, Sven Muller, Susanne Moller, Andreas Schonau, Eva Balslev, Bent EjlertsenAbstract:Copy number changes in TOP2A have frequently been linked to ERBB2 (HER2) amplified breast cancers. To study this relationship, copy number changes of ERBB2 and TOP2A were investigated by fluorescence in situ hybridization (FISH) in two cell lines; one characterized by having amplification of both genes and the other by having amplification of ERBB2 and deletion of TOP2A. The characteristics are compared to findings on paired ERBB2 and TOP2A data from 649 patients with invasive breast cancer from a previously published biomarker study. The physical localization of FISH signals in metaphase spreads from cell lines showed that simultaneous amplification is not a simple co-amplification of a whole amplicon containing both genes. Most gene signals are translocated to abnormal marker chromosomes. ERBB2 genes but not TOP2A genes are present in tandem amplicons, leading to a higher ERBB2 ratio. This observation was confirmed by patient FISH data: among 276 (43% of all patients) abnormal tumors, 67% had different ERBB2 and TOP2A status. ERBB2 amplification with normal TOP2A status was found in 36% of the abnormal tumors (15% of all patients). Simultaneous amplification of both genes was found in 28% of the abnormal tumors (12% of all patients) while TOP2A deletion and ERBB2 amplification was observed in 16% of the abnormal cases (8% of all patients). A small number of tumors had TOP2A amplification (4%) or deletion (6%) without simultaneous changes of the ERBB2 gene. ERBB2 deletion was also observed (5%) but only in tumors with simultaneous TOP2A deletion. The average gene/reference ratio was significantly different: 5.0 for TOP2A but 7.2 for ERBB2 in the amplified tumors (P<0.01). Amplification of the two genes may be caused by different mechanisms, leading to higher level of amplification for ERBB2 compared to TOP2A. In the majority of breast cancer patients, simultaneous aberration of ERBB2 and TOP2A is not explained by simple co-amplification.
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Aberrations of HER2 and TOP2A Genes in Breast Cancer.
Poster Session Abstracts, 2009Co-Authors: Kirsten Vang Nielsen, Ann Knoop, Sven Muller, Susanne Moller, Andreas Schonau, Eva Balslev, Bent EjlertsenAbstract:Copy number changes in TOP2A are frequently observed in HER2 amplified breast cancers, and amplification of a whole amplicon containing both genes has been suggested as the underling mechanism. Here, we describe copy number changes of HER2 and TOP2A in two cell lines; one characterized by having amplification of both genes and the other by having amplification of HER2 and deletion of TOP2A. The characteristics are compared to findings in patients with invasive breast cancer.Material and methods: Fluorescence in situ hybridization (FISH) with HER2, TOP2A and centromere 17 (CEN-17) probes was performed on metaphases, interphases and cut sections from breast cancer cell lines. Paired HER2/CEN-17 and TOP2A/CEN-17 data was available from 649 patients from a previously published biomarker study (Knoop et al. J Clin Oncol 2005;23:7483-90).Results: The physical localization of FISH signals in metaphase spreads from cell lines showed that simultaneous amplification is not a simple co-amplification of a whole amplicon containing both genes. HER2 and TOP2A aberrations seem to be due to different mechanisms of amplifications. Most gene signals are translocated to abnormal marker chromosomes. HER2 genes but not TOP2A genes are present in tandem amplicons, leading to a higher HER2 ratio. This observation was confirmed by patient FISH data: 373 tumors (57%) had normal status for both genes. Among the 276 abnormal tumors, 67% had different HER2 and TOP2A status. Simultaneous amplification of both genes was found in 28% of the abnormal tumors (12% of all patients) while deletion of both genes was observed in 5% of the cases (2% of all patients). The average gene/reference ratio was significantly different: 5.0 for TOP2A but 7.2 for HER2 in the amplified tumors (P Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2119.
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Aberrations of ERBB2 and TOP2A genes in breast cancer.
Molecular oncology, 2009Co-Authors: Kirsten Vang Nielsen, Ann Knoop, Sven Muller, Susanne Moller, Andreas Schonau, Eva Balslev, Bent EjlertsenAbstract:Copy number changes in TOP2A have frequently been linked to ERBB2 (HER2) amplified breast cancers. To study this relationship, copy number changes of ERBB2 and TOP2A were investigated by fluorescence in situ hybridization (FISH) in two cell lines; one characterized by having amplification of both genes and the other by having amplification of ERBB2 and deletion of TOP2A. The characteristics are compared to findings on paired ERBB2 and TOP2A data from 649 patients with invasive breast cancer from a previously published biomarker study. The physical localization of FISH signals in metaphase spreads from cell lines showed that simultaneous amplification is not a simple co-amplification of a whole amplicon containing both genes. Most gene signals are translocated to abnormal marker chromosomes. ERBB2 genes but not TOP2A genes are present in tandem amplicons, leading to a higher ERBB2 ratio. This observation was confirmed by patient FISH data: among 276 (43% of all patients) abnormal tumors, 67% had different ERBB2 and TOP2A status. ERBB2 amplification with normal TOP2A status was found in 36% of the abnormal tumors (15% of all patients). Simultaneous amplification of both genes was found in 28% of the abnormal tumors (12% of all patients) while TOP2A deletion and ERBB2 amplification was observed in 16% of the abnormal cases (8% of all patients). A small number of tumors had TOP2A amplification (4%) or deletion (6%) without simultaneous changes of the ERBB2 gene. ERBB2 deletion was also observed (5%) but only in tumors with simultaneous TOP2A deletion. The average gene/reference ratio was significantly different: 5.0 for TOP2A but 7.2 for ERBB2 in the amplified tumors (P
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a meta analysis of phase iii trials evaluating the predictive value of her2 and topoisomerase ii alpha in early breast cancer patients treated with cmf or anthracycline based adjuvant therapy
Cancer Research, 2009Co-Authors: Jorma Isola, Bent Ejlertsen, Christine Desmedt, Denis Larsimont, Fanny Piette, Kathleen I Pritchard, John M S Bartlett, Minna Tanner, H Mouridsen, Frances P OmalleyAbstract:CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #705 Background: Retrospective studies have suggested that HER2 and topoisomerase II α (TOP2A) might predict sensitivity to anthracyclines (A) Methods: Four phase III trials comparing A with CMF in early breast cancer (EBC) patients (pts), and with available primary tumor samples, were identified. HER2 and TOP2A genes were evaluated locally by FISH (amplification if ratio ≥ 2). Data were centralized at the statistical office (IDDI, Belgium). On-site visits were performed to check data quality and laboratory procedures. HER2 and TOP2A local scores were validated by submitting randomly selected samples to a central lab (CL) (University of Tampere – Finland), for a 3 color FISH test (HER2, TOP2A, centromere 17, Abbott Labs, IL, USA). Estrogen (ER), progesterone (PgR) receptors, and grade (G) were evaluated locally (no score validation at the CL). Results: We present the results of the planned interim analysis on 1944 pts. Final results (± 3500 pts) will be presented when tumor sample collection for the UK trial is complete. HER2 local scores were validated at the CL in 137 cases (discordance: 8/137, 5.8%). TOP2A local scores were validated in 123 cases (discordance 38/123, 30.8%). Half of the TOP2A discordant cases were locally deleted-centrally normal or vice versa. ![][1] A planned exploratory analysis evaluated the TOP2A predictivity in 4 biologically homogeneous groups: highly or moderately hormone sensitive, HER2+ and ER/PgR negative, triple negative (TN). This analysis did not enhance the TOP2A predictivity in any of the 4 groups. In the TN group (294 pts), the DFS HR was 0.77 (0.54-1.09), suggesting that benefit from A might not be confined to HER2+ pts. Conclusions: The interim analysis shows that HER2 and TOP2A have a clinically modest and a statistically borderline predictive value. In triple negative disease A may be superior to CMF. Acknowledgments: Abbott Laboratories, Belgian Federation Against Cancer, Cancer Research UK, Les Amis de l'Institut Bordet, Pfizer, Scottish BC trials group. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 705. [1]: /embed/graphic-1.gif
Ross F Collery - One of the best experts on this subject based on the ideXlab platform.
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Maternal topoisomerase II alpha, not topoisomerase II beta, enables embryonic development of zebrafish TOP2A-/- mutants
BMC developmental biology, 2011Co-Authors: B. Sapetto-rebow, Sarah Mcloughlin, Lynne C. O'shea, Olivia O'leary, Jason R. Willer, Yolanda Alvarez, Ross F Collery, Jacintha O'sullivan, Freek Van Eeden, Carmel HenseyAbstract:Background Genetic alterations in human topoisomerase II alpha (TOP2A) are linked to cancer susceptibility. TOP2A decatenates chromosomes and thus is necessary for multiple aspects of cell division including DNA replication, chromosome condensation and segregation. Topoisomerase II alpha is also required for embryonic development in mammals, as mouse TOP2A knockouts result in embryonic lethality as early as the 4-8 cell stage. The purpose of this study was to determine whether the extended developmental capability of zebrafish TOP2A mutants arises from maternal expression of TOP2A or compensation from its top2b paralogue.
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maternal topoisomerase ii alpha not topoisomerase ii beta enables embryonic development of zebrafish TOP2A mutants
BMC Developmental Biology, 2011Co-Authors: B Sapettorebow, Sarah Mcloughlin, Jason R. Willer, Yolanda Alvarez, Ross F Collery, Lynne C Oshea, Olivia Oleary, Jacintha OsullivanAbstract:Background Genetic alterations in human topoisomerase II alpha (TOP2A) are linked to cancer susceptibility. TOP2A decatenates chromosomes and thus is necessary for multiple aspects of cell division including DNA replication, chromosome condensation and segregation. Topoisomerase II alpha is also required for embryonic development in mammals, as mouse TOP2A knockouts result in embryonic lethality as early as the 4-8 cell stage. The purpose of this study was to determine whether the extended developmental capability of zebrafish TOP2A mutants arises from maternal expression of TOP2A or compensation from its top2b paralogue.
Sarah Mcloughlin - One of the best experts on this subject based on the ideXlab platform.
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Maternal topoisomerase II alpha, not topoisomerase II beta, enables embryonic development of zebrafish TOP2A-/- mutants
BMC developmental biology, 2011Co-Authors: B. Sapetto-rebow, Sarah Mcloughlin, Lynne C. O'shea, Olivia O'leary, Jason R. Willer, Yolanda Alvarez, Ross F Collery, Jacintha O'sullivan, Freek Van Eeden, Carmel HenseyAbstract:Background Genetic alterations in human topoisomerase II alpha (TOP2A) are linked to cancer susceptibility. TOP2A decatenates chromosomes and thus is necessary for multiple aspects of cell division including DNA replication, chromosome condensation and segregation. Topoisomerase II alpha is also required for embryonic development in mammals, as mouse TOP2A knockouts result in embryonic lethality as early as the 4-8 cell stage. The purpose of this study was to determine whether the extended developmental capability of zebrafish TOP2A mutants arises from maternal expression of TOP2A or compensation from its top2b paralogue.
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maternal topoisomerase ii alpha not topoisomerase ii beta enables embryonic development of zebrafish TOP2A mutants
BMC Developmental Biology, 2011Co-Authors: B Sapettorebow, Sarah Mcloughlin, Jason R. Willer, Yolanda Alvarez, Ross F Collery, Lynne C Oshea, Olivia Oleary, Jacintha OsullivanAbstract:Background Genetic alterations in human topoisomerase II alpha (TOP2A) are linked to cancer susceptibility. TOP2A decatenates chromosomes and thus is necessary for multiple aspects of cell division including DNA replication, chromosome condensation and segregation. Topoisomerase II alpha is also required for embryonic development in mammals, as mouse TOP2A knockouts result in embryonic lethality as early as the 4-8 cell stage. The purpose of this study was to determine whether the extended developmental capability of zebrafish TOP2A mutants arises from maternal expression of TOP2A or compensation from its top2b paralogue.
Ann Knoop - One of the best experts on this subject based on the ideXlab platform.
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CEF is superior to CMF for tumours with TOP2A aberrations: a Subpopulation Treatment Effect Pattern Plot (STEPP) analysis on Danish Breast Cancer Cooperative Group Study 89D.
Breast cancer research and treatment, 2010Co-Authors: Katrín Á Gunnarsdóttir, Maj-britt Jensen, David Zahrieh, Richard D. Gelber, Ann Knoop, Marco Bonetti, Henning T. Mouridsen, Bent EjlertsenAbstract:The aim of this study was to examine TOP2A gene copy number changes as a means to identify groups of breast cancer patients with superior benefit from treatment with anthracyclines. Tumour tissue was retrospectively collected and successfully analysed for TOP2A in 773 of 980 Danish patients randomly assigned to receive intravenous CMF (cyclophosphamide, methotrexate and fluorouracil) or CEF (cyclophosphamide, epirubicin and fluorouracil) in DBCG trial 89D. Subgroup analyses on this material published by Knoop et al. (J Clin Oncol 23:7483–7490, 2005) and updated by Nielsen et al. (Acta Oncol 47:725–734, 2008) demonstrated that superiority of CEF over CMF is limited to patients with TOP2A aberrations, defined as patients whose tumours have TOP2A ratio below 0.8 or above 2.0. The Subpopulation Treatment Effect Pattern Plot (STEPP) technique was applied to these data to explore the pattern of treatment effect relative to TOP2A and to compare that pattern to the ranges previously used to define ‘aberrations’. The pattern of treatment effect illustrated by the STEPP analysis confirmed that the superiority of CEF over CMF is indeed limited to patients whose tumours have high or low TOP2A ratios. The hypothesis of no treatment effect–covariate interaction was rejected (P = 0.02). Furthermore, results indicated that the interval of TOP2A ratios hitherto denoted as ‘normal’ could be narrower than previously assumed. A more optimal separation of TOP2A subgroups could be obtained by altering cut-points currently used to define TOP2A amplified and TOP2A deleted tumours by narrowing the TOP2A normal interval, and consequently enlarging the population with TOP2A aberrated tumours.
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aberrations of erbb2 and TOP2A genes in breast cancer
Molecular Oncology, 2010Co-Authors: Kirsten Vang Nielsen, Ann Knoop, Sven Muller, Susanne Moller, Andreas Schonau, Eva Balslev, Bent EjlertsenAbstract:Copy number changes in TOP2A have frequently been linked to ERBB2 (HER2) amplified breast cancers. To study this relationship, copy number changes of ERBB2 and TOP2A were investigated by fluorescence in situ hybridization (FISH) in two cell lines; one characterized by having amplification of both genes and the other by having amplification of ERBB2 and deletion of TOP2A. The characteristics are compared to findings on paired ERBB2 and TOP2A data from 649 patients with invasive breast cancer from a previously published biomarker study. The physical localization of FISH signals in metaphase spreads from cell lines showed that simultaneous amplification is not a simple co-amplification of a whole amplicon containing both genes. Most gene signals are translocated to abnormal marker chromosomes. ERBB2 genes but not TOP2A genes are present in tandem amplicons, leading to a higher ERBB2 ratio. This observation was confirmed by patient FISH data: among 276 (43% of all patients) abnormal tumors, 67% had different ERBB2 and TOP2A status. ERBB2 amplification with normal TOP2A status was found in 36% of the abnormal tumors (15% of all patients). Simultaneous amplification of both genes was found in 28% of the abnormal tumors (12% of all patients) while TOP2A deletion and ERBB2 amplification was observed in 16% of the abnormal cases (8% of all patients). A small number of tumors had TOP2A amplification (4%) or deletion (6%) without simultaneous changes of the ERBB2 gene. ERBB2 deletion was also observed (5%) but only in tumors with simultaneous TOP2A deletion. The average gene/reference ratio was significantly different: 5.0 for TOP2A but 7.2 for ERBB2 in the amplified tumors (P<0.01). Amplification of the two genes may be caused by different mechanisms, leading to higher level of amplification for ERBB2 compared to TOP2A. In the majority of breast cancer patients, simultaneous aberration of ERBB2 and TOP2A is not explained by simple co-amplification.
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Aberrations of HER2 and TOP2A Genes in Breast Cancer.
Poster Session Abstracts, 2009Co-Authors: Kirsten Vang Nielsen, Ann Knoop, Sven Muller, Susanne Moller, Andreas Schonau, Eva Balslev, Bent EjlertsenAbstract:Copy number changes in TOP2A are frequently observed in HER2 amplified breast cancers, and amplification of a whole amplicon containing both genes has been suggested as the underling mechanism. Here, we describe copy number changes of HER2 and TOP2A in two cell lines; one characterized by having amplification of both genes and the other by having amplification of HER2 and deletion of TOP2A. The characteristics are compared to findings in patients with invasive breast cancer.Material and methods: Fluorescence in situ hybridization (FISH) with HER2, TOP2A and centromere 17 (CEN-17) probes was performed on metaphases, interphases and cut sections from breast cancer cell lines. Paired HER2/CEN-17 and TOP2A/CEN-17 data was available from 649 patients from a previously published biomarker study (Knoop et al. J Clin Oncol 2005;23:7483-90).Results: The physical localization of FISH signals in metaphase spreads from cell lines showed that simultaneous amplification is not a simple co-amplification of a whole amplicon containing both genes. HER2 and TOP2A aberrations seem to be due to different mechanisms of amplifications. Most gene signals are translocated to abnormal marker chromosomes. HER2 genes but not TOP2A genes are present in tandem amplicons, leading to a higher HER2 ratio. This observation was confirmed by patient FISH data: 373 tumors (57%) had normal status for both genes. Among the 276 abnormal tumors, 67% had different HER2 and TOP2A status. Simultaneous amplification of both genes was found in 28% of the abnormal tumors (12% of all patients) while deletion of both genes was observed in 5% of the cases (2% of all patients). The average gene/reference ratio was significantly different: 5.0 for TOP2A but 7.2 for HER2 in the amplified tumors (P Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2119.
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Aberrations of ERBB2 and TOP2A genes in breast cancer.
Molecular oncology, 2009Co-Authors: Kirsten Vang Nielsen, Ann Knoop, Sven Muller, Susanne Moller, Andreas Schonau, Eva Balslev, Bent EjlertsenAbstract:Copy number changes in TOP2A have frequently been linked to ERBB2 (HER2) amplified breast cancers. To study this relationship, copy number changes of ERBB2 and TOP2A were investigated by fluorescence in situ hybridization (FISH) in two cell lines; one characterized by having amplification of both genes and the other by having amplification of ERBB2 and deletion of TOP2A. The characteristics are compared to findings on paired ERBB2 and TOP2A data from 649 patients with invasive breast cancer from a previously published biomarker study. The physical localization of FISH signals in metaphase spreads from cell lines showed that simultaneous amplification is not a simple co-amplification of a whole amplicon containing both genes. Most gene signals are translocated to abnormal marker chromosomes. ERBB2 genes but not TOP2A genes are present in tandem amplicons, leading to a higher ERBB2 ratio. This observation was confirmed by patient FISH data: among 276 (43% of all patients) abnormal tumors, 67% had different ERBB2 and TOP2A status. ERBB2 amplification with normal TOP2A status was found in 36% of the abnormal tumors (15% of all patients). Simultaneous amplification of both genes was found in 28% of the abnormal tumors (12% of all patients) while TOP2A deletion and ERBB2 amplification was observed in 16% of the abnormal cases (8% of all patients). A small number of tumors had TOP2A amplification (4%) or deletion (6%) without simultaneous changes of the ERBB2 gene. ERBB2 deletion was also observed (5%) but only in tumors with simultaneous TOP2A deletion. The average gene/reference ratio was significantly different: 5.0 for TOP2A but 7.2 for ERBB2 in the amplified tumors (P
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The value of TOP2A gene copy number variation as a biomarker in breast cancer: Update of DBCG trial 89D.
Acta oncologica (Stockholm Sweden), 2008Co-Authors: Kirsten Vang Nielsen, Ann Knoop, Bent Ejlertsen, Susanne Moller, Helle Knudsen, Jan Trøst Jørgensen, Henning T. MouridsenAbstract:Background. Previous analyses of TOP2A and HER2 in the Danish Breast Cancer Coopererative Group (DBCG) trial 89D suggested that TOP2A amplifications and possible also deletions are predictive markers for the effect of adjuvant epirubicin in patients with primary breast cancer. We present an updated and extended statistical analysis, requested for IVD-labeling of TOP2A testing. Material and methods. In the DBCG trial 89D 980 Danish patients were randomly assigned to nine cycles of intravenous CMF (cyclophosphamide, methotrexate, and fluorouracil) or CEF (cyclophosphamide, epirubicin, and fluorouracil). Archival tumor tissue was collected retrospectively from 806 of these patients in a prospectively designed, biological sub-study, and was successfully analyzed for TOP2A aberrations and HER2 status in 773 samples (96%). Recurrence-free survival (RFS) was the primary endpoint. Results. TOP2A aberrations (amplifications and deletions) were significantly associated with shorter RFS (p
Yves Pommier - One of the best experts on this subject based on the ideXlab platform.
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proteolytic degradation of topoisomerase ii top2 enables the processing of top2 dna and top2 rna covalent complexes by tyrosyl dna phosphodiesterase 2 tdp2
Journal of Biological Chemistry, 2014Co-Authors: Matthew J Schellenberg, Christophe Marchand, John L. Nitiss, Shar Yin N Huang, Monica Abdelmalak, Karin C Nitiss, Scott R Williams, Yves PommierAbstract:Eukaryotic type II topoisomerases (Top2α and Top2β) are homodimeric enzymes; they are essential for altering DNA topology by the formation of normally transient double strand DNA cleavage. Anticancer drugs (etoposide, doxorubicin, and mitoxantrone) and also Top2 oxidation and DNA helical alterations cause potentially irreversible Top2·DNA cleavage complexes (Top2cc), leading to Top2-linked DNA breaks. Top2cc are the therapeutic mechanism for killing cancer cells. Yet Top2cc can also generate recombination, translocations, and apoptosis in normal cells. The Top2 protein-DNA covalent complexes are excised (in part) by tyrosyl-DNA-phosphodiesterase 2 (TDP2/TTRAP/EAP2/VPg unlinkase). In this study, we show that irreversible Top2cc induced in suicidal substrates are not processed by TDP2 unless they first undergo proteolytic processing or denaturation. We also demonstrate that TDP2 is most efficient when the DNA attached to the tyrosyl is in a single-stranded configuration and that TDP2 can efficiently remove a tyrosine linked to a single misincorporated ribonucleotide or to polyribonucleotides, which expands the TDP2 catalytic profile with RNA substrates. The 1.6-Å resolution crystal structure of TDP2 bound to a substrate bearing a 5′-ribonucleotide defines a mechanism through which RNA can be accommodated in the TDP2 active site, albeit in a strained conformation.
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DNA topoisomerases and their poisoning by anticancer and antibacterial drugs.
Chemistry & Biology, 2010Co-Authors: Yves Pommier, Hongliang Zhang, Elisabetta Leo, Christophe MarchandAbstract:DNA topoisomerases are the targets of important anticancer and antibacterial drugs. Camptothecins and novel noncamptothecins in clinical development (indenoisoquinolines and ARC-111) target eukaryotic type IB topoisomerases (Top1), whereas human type IIA topoisomerases (Top2α and Top2β) are the targets of the widely used anticancer agents etoposide, anthracyclines (doxorubicin, daunorubicin), and mitoxantrone. Bacterial type II topoisomerases (gyrase and Topo IV) are the targets of quinolones and aminocoumarin antibiotics. This review focuses on the molecular and biochemical characteristics of topoisomerases and their inhibitors. We also discuss the common mechanism of action of topoisomerase poisons by interfacial inhibition and trapping of topoisomerase cleavage complexes.
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dna topoisomerase i inhibitors chemistry biology and interfacial inhibition
Chemical Reviews, 2009Co-Authors: Yves PommierAbstract:DNA topoisomerases I and II (Top1 and Top2) are established molecular targets of anticancer drugs.1–5 Mammalian somatic cells express six topoisomerase genes: two TOP1 (TOP1 and TOP1mt), two TOP2 (TOP2α and β), two topoisomerase III (TOP3α and β)6,7 (Figure 1A). The most recently discovered eukaryotic topoisomerase is mitochondrial Top1 (Top1mt), which we reported in 2001.8,9 Figure 1 Schematic architecture of the topoisomerase cleavage complexes A common feature of topoisomerases is their catalytic mechanism, which in all cases consists in a nucleophilic attack of a DNA phosphodiester bond by a catalytic tyrosyl residue from the topoisomerase. The resulting covalent attachment of the tyrosine to the DNA phosphate is either at the 3′-end of the broken DNA in the case of Top1 enzymes (Top1 and Top1mt) or at the 5′-end of the broken DNA for the other topoisomerases (Figure 1). Thus, Top1 enzymes are the only topoisomerases that form a covalent link with the 3′-end of the broken DNA while generating a 5′-hydroxyl end at the other end of the break. In that respect, the eukaryotic Top1 enzymes belong to the broader family of site-specific tyrosine recombinases of prokaryotes and yeast (e.g., XerCD of Escherichia coli, bacteriophage λ integrase and Cre recombinase, and Flp of Saccharomyces cerevisiae). Another unique feature of the Top1 enzymes is their DNA relaxation mechanism by “controlled rotation” rather than by “strand passage”.10–12 In other words, Top1 enzymes relax DNA by letting the 5′-hydroxyl end swivel around the intact strand. This processive reaction does not require ATP or divalent metal binding, which is different from Top2 enzymes, which require both ATP hydrolysis and Mg2+.5,13 Top3 enzymes, which, like other type IA topoisomerases require Mg2+ (but no ATP) for catalysis14 are not very active in relaxing DNA supercoiling. They can relax DNA when it is very negatively supercoiled (single-stranded) one turn at a time.15 Moreover, both Top2 and Top3 enzymes change DNA topology by a strand passage distributive mechanism rather than by the processive controlled rotation of the Top1 enzymes. In the case of the Top2 enzymes, a full DNA duplex [referred to as the T (transported) strand] goes through the double-strand break made by an enzyme homodimer5,16,17 (Figure 1A). In the case of the Top3 enzymes, a single strand goes through the single-stranded break,14 typically at double-Holliday junction crossovers.18 The remarkable efficiency of the nicking-closing activity of Top1 enables the enzyme to relax both negatively and positively supercoiled DNA (even at 0°C)19 with similar efficiency.12 This is in contrast with Top2α, which relaxes more efficiently positive supercoiling.20 Of note, Top2β, like Top1 relaxes both positive and negative supercoils similarly.20 Removing positive supercoils is required for replication and transcription progression. Otherwise their accumulation in advance of replication and transcription complexes hinders the melting of the DNA duplex (by helicases) and consequently polymerase translocation along the DNA template. The normal nicking-closing activity of Top1 can however be uncoupled when the 5′-hydroxyl end generated by the nicking reaction becomes misaligned; for instance at preexisting base lesions or DNA nicks.21,22 In such cases, the Top1 cleavage complex (Top1cc) remains without effective legitimate religation partner. Those Top1-DNA covalent complexes are commonly referred to as “suicide complexes”. Under such conditions, Top1 can nevertheless religate an illegitimate (“foreign”) 5-hydroxyl-DNA end and act as a recombinase.23 This property is routinely used for molecular cloning (TOPO® Cloning, Invitrogen) using vaccinia Top1.24
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the iron chelator dp44mt causes dna damage and selective inhibition of topoisomerase iiα in breast cancer cells
Cancer Research, 2009Co-Authors: Ashutosh V Rao, Yves Pommier, Sarah R Klein, Keli Agama, Eriko Toyoda, Noritaka Adachi, Emily ShacterAbstract:Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is being developed as an iron chelator with selective anticancer activity. We investigated the mechanism whereby Dp44mT kills breast cancer cells, both as a single agent and in combination with doxorubicin. Dp44mT alone induced selective cell killing in the breast cancer cell line MDA-MB-231 when compared with healthy mammary epithelial cells (MCF-12A). It induces G1 cell cycle arrest and reduces cancer cell clonogenic growth at nanomolar concentrations. Dp44mT, but not the iron chelator desferal, induces DNA double-strand breaks quantified as S139 phosphorylated histone foci (γ-H2AX) and Comet tails induced in MDA-MB-231 cells. Doxorubicin-induced cytotoxicity and DNA damage were both enhanced significantly in the presence of low concentrations of Dp44mT. The chelator caused selective poisoning of DNA topoisomerase IIα (top2α) as measured by an in vitro DNA cleavage assay and cellular topoisomerase-DNA complex formation. Heterozygous Nalm-6 top2α knockout cells (top2α+/−) were partially resistant to Dp44mT-induced cytotoxicity compared with isogenic top2α+/+ or top2β−/− cells. Specificity for top2α was confirmed using top2α and top2β small interfering RNA knockdown in HeLa cells. The results show that Dp44mT is cytotoxic to breast cancer cells, at least in part, due to selective inhibition of top2α. Thus, Dp44mT may serve as a mechanistically unique treatment for cancer due to its dual ability to chelate iron and inhibit top2α activity. [Cancer Res 2009;69(3):948–57]