Human DNA topoisomerase I: An anticancer drug target present in human sarcomas

doi:10.1053/hupa.2002.124911

Human Pathology

Volume 33, Issue 6, June 2002, Pages 599-607

https://doi.org/10.1053/hupa.2002.124911 Get rights and content

Abstract

New anticancer drugs targeting DNA topoisomerase I (topo I) are showing activity against human sarcomas. Laboratory studies have indicated that cells responsive to topo I–targeted drugs have elevated levels of topo I, require active DNA replication, and may require a functional apoptotic pathway. In this study, we evaluated these potential markers of topo I–targeted drug sensitivity in 55 cases of human sarcoma (42 high grade, 4 intermediate grade, and 9 low grade). By immunohistochemical staining, we observed elevated topo I expression in 20 of 55 neoplasms (36%). Immunohistochemical staining for the proliferation marker DNA topoisomerase II-alpha (topo II-alpha), showed that 15 of 55 neoplasms (27%) had topo II-alpha indices >50, indicating a large number of actively cycling tumor cells. Abnormal p53 expression was observed in 19 of the 55 cases (35%). None of the cases were interpreted as positive for ALK-1. To complement our immunohistochemical staining of topo I, we isolated functionally active topo I from extracts of a human sarcoma. These isolates demonstrated that sarcoma topo I is sensitive to topo I–targeted anticancer drugs. Of the 55 cases of human sarcoma, 7 (13%) had high levels of topo I, a large number of cycling tumor cells, and normal p53 expression. These are the molecular parameters that might suggest responsiveness to drugs targeting topo I. HUM PATHOL 33:599-607. Copyright 2002, Elsevier Science (USA). All rights reserved.

Section snippets

Sources of tissue

Paraffin blocks from 55 sarcoma cases were retrieved from the surgical pathology files at the University of Utah Health Sciences Center. All cases were reviewed to confirm the diagnosis. Forty-two of the tumors were high grade, 4 were intermediate grade, and 9 were low grade. The sarcomas were graded as low, intermediate, or high according to tumor differentiation, necrosis, and mitotic count.²⁵

Chemical and antibodies

The topo I antibody is a mouse monoclonal antibody that recognizes human topo I on Western blots and

Patient characteristics

The clinicopathologic characteristics of the sarcoma patients in our study are shown in Table 1.The age of the patients ranged from 19 to 93 years with an average age of 56.1 years. Nine neoplasms were low grade, 4 were intermediate grade, and 42 were high grade. These sarcomas all originated from the soft tissues and were represented by 20 cases of malignant fibrous histiocytoma, 20 cases of undifferentiated/spindle cell sarcoma, 8 cases of fibrosarcoma, 4 cases of leiomyosarcoma, 2 cases of

Discussion

Topo I inhibitors are showing activity against a wide range of malignancies. Several topo I–targeted drugs have been approved for the treatment of specific solid tumors. Despite these advances, many human neoplasms are resistant to current treatment regimens. Among these resistant malignancies are human sarcomas. They are difficult to treat, and the current medical treatment options are limited both in number and efficacy.4, 5

In this study, we evaluated a series of 55 human sarcomas for

Acknowledgements

The authors thank J. Chris Pitchford for his ongoing interest in the topoisomerases as anticancer drug targets.

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MET increases the sensitivity of gefitinib-resistant cells to SN-38, an active metabolite of irinotecan, by Up-regulating the topoisomerase i activity
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In PC-9/Met cells, the mRNA and protein expression, and the activity of topo I were higher than in the PC-9 cells. The results of the topo I activity assays performed in this study are consistent with previous studies showing that the sensitivity to topo I inhibitors correlates with the topo I activity of the tumor cells.25–28 We next examined the effects of MET-specific siRNA or MET stimulation by HGF on the topo I level in these experiments.
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The sensitivity of PC-9 and PC-9/Met to SN-38 was assessed by performing water soluble tetrazolium salt (WST-1) assays. Topoisomerase I (topo I) activities were determined for the cell lines cultured in the presence of hepatocyte growth factor and for those of which MET expression was knocked down by introducing a MET-specific small interfering RNA.
PC-9/Met exhibited higher topo I activities, and higher topo I gene and protein expression levels than PC-9 did. Suppression of MET expression by a MET-specific small interfering RNA led to a decrease in the topo I protein expression in the PC-9/Met cells. The stimulation of PC-9 with hepatocyte growth factor caused an increase in the topo I protein level via the activation of MET.
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Topoisomerase I (TopoI), an essential enzyme, produces a DNA single strand break allowing DNA relaxation for replication. The enzymatic mechanism involves sequential transesterifcations. The breakage and closure reactions generate phosphodiester bonds and similar free energies, so the reaction is freely reversible. The TopoI reaction intermediate consists of enzyme covalently linked to DNA dubbed a ‘cleavable complex’. Covalently bound TopoI–DNA complexes can be recovered. Camptothecin analogs, topotecan and irinotecan, are approved TopoI-targeted drugs. Both have limitations due to the equilibrium between the camptothecin lactone and ring-opened forms. Several strategies are being explored to develop improved TopoI inhibitors. Homocamptothecins, in which the metabolically labile camptothecin lactone is replaced with a more stable seven-membered β-hydroxylactone, are potent anticancer agents. Gimatecan is a seven-position modified lipophilic camptothecin developed to provide rapid uptake and accumulation in cells and a stable TopoI–DNA–drug ternary complex. Diflomotecan, a homocamptothecin, and gimatecan are in Phase II clinical trial. Among non-camptothecins, edotecarin, an indolocarbazole that results in DNA C/T-G cleavage compared with T-G/A for camptothecins, is in Phase II clinical trial. Indenoisoquinolines were identified as TopoI inhibitors by the NCI 60-cell line COMPARE analysis. Co-crystal structures of two indenoisoquinolines with TopoI–DNA elucidated the structure of the ternary complex. Indenoisoquinolines are in preclinical development. Dibenzonaphthyridinone TopoI inhibitors have undergone extensive structure–activity examination. ARC-111 was selected for in-depth preclinical study. Biomarkers are under investigation to predict clinical efficacy from preclinical models, to allow determination of drug targeting in vivo and to aid selection of patients most likely to benefit from TopoI inhibitor therapy. γ-H2AX formation may be a useful pharmacodynamic marker. A gene signature developed for topotecan sensitivity/resistance may have value in patient identification. Convergence of these efforts should result in clinically effective second generation TopoI inhibitors.
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^*: Supported by the ARUP Institute for Clinical and Experimental Pathology, the Biotechnological and Biological Sciences Research Council (UK), and Pharmacia Upjohn Inc.

^**: Address correspondence and reprint requests to Joseph A. Holden, MD, PHD, University of Utah Health Sciences Center, Salt Lake City, UT 84132.

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Original ContributionsHuman DNA topoisomerase I: An anticancer drug target present in human sarcomas*,**

Abstract

Section snippets

Sources of tissue

Chemical and antibodies

Patient characteristics

Discussion

Acknowledgements

Biochim Biophys Acta

Ann Oncol

Cell

Biochem Biophys Acta

J Biol Chem