DNA Topoisomerase I-Targeting Drugs as Radiation Sensitizers

OncologyONCOLOGY Vol 13 No 10
Volume 13
Issue 10

Combination chemoradiation, alone or as an adjuvant to surgery, has been shown to improve treatment outcomes in a number of human malignancies, but may be limited by normal tissue toxicities. A primary challenge in

ABSTRACT: Combination chemoradiation, alone or as an adjuvant to surgery, has been shown to improve treatment outcomes in a number of human malignancies, but may be limited by normal tissue toxicities. A primary challenge in radiation oncology is the development of drugs that can selectively enhance the cytotoxicity of ionizing radiation against tumor cells. Mammalian DNA topoisomerase I is the major cytotoxic target of a number of newly developed anticancer drugs that have shown efficacy against solid tumors, including colon cancer, ovarian cancer, lung cancer, cancer of the head and neck, and pediatric cancers. Topoisomerase I-targeting drugs exert their cytotoxic effect by producing enzyme-mediated DNA damage, rather than by directly inhibiting enzyme catalytic activity. DNA topoisomerase I recently has been established as a biochemical mediator of radiosensitization in cultured mammalian cells by camptothecin derivatives. Interestingly, this sensitization appears to be schedule-dependent, cell cycle phase-specific, cell line-dependent, and not strictly dependent on drug cytotoxicity. Clinical chemoradiation trials using camptothecin derivatives are currently ongoing. Future studies aimed at better understanding the underlying mechanisms of molecular radiosensitization with topoisomerase I-targeting drugs are pivotal to the clinical application of these agents, as well as in guiding the development of more effective radiosensitizers. [ONCOLOGY 13(Suppl 5):39-46,1999]


Combined-modality therapy with chemotherapy and radiation therapy has gained increasing importance in the treatment of human solid tumors.[1,2] In addition to controlling potential or overt metastatic disease, a number of chemotherapeutic drugs also enhance the cytotoxic effect of ionizing radiation [3,4] and help locally control the primary disease. However, the efficacy and use of currently available chemoradiation regimens are limited by their toxicities to normal tissue. The development of more effective chemoradiation regimens using radiosensitizers, which can preferentially enhance the cytotoxicity of ionizing radiation in cancer cells, remains a major challenge in radiation oncology.

DNA topoisomerase I is a novel cellular target of a number of antineoplastic compounds,[5] including the camptothecin derivatives[6-9] and the newly identified DNA minor groove-binding ligands (MGBLs) such as Hoechst 33342.[10,11] Drug interference with topoisomerase I-mediated cleavage rejoining of DNA strands is thought to be the common mechanism of action of these drugs.[6-11] Instead of direct inhibition of catalytic activity of topoisomerase I, topoisomerase I drugs produce their cytotoxic effects by converting an essential DNA topology-modifying activity into a DNA-breaking poison, which damages DNA through interactions with cellular processes such as DNA replication.[5,12-15] The presence of up-regulated levels of topoisomerase I in tumor cells compared to normal cells suggests a therapeutic advantage of topoisomerase I-targeting drugs selective against slow-growing as well as rapidly proliferating tumors.[16-18] Recent advances in the molecular mechanisms of cytotoxic action and radiosensitization of DNA topoisomerase I-targeting drugs offer a unique opportunity for developing more effective chemoradiation therapy against human cancers.

DNA Topoisomerase I is an Essential Nuclear Enzyme

Only one type-I human DNA topoisomerase has been identified as a molecular target of anticancer drugs. The human topoisomerase I, encoded by a single-copy TOP1 gene on chromosome 20, is a monomeric 100 kDa protein. It relaxes both positively and negatively supercoiled DNA and requires no energy cofactor for its activity.[19-21] The activities of topoisomerase I are important for many aspects of DNA metabolism, including the initiation and elongation of RNA transcription, DNA replication, and the regulation of DNA supercoiling, which is essential for maintaining the stability of the genome.[19-21]

During a typical catalytic cycle, topoisomerase I cleaves the DNA backbone, allowing the passage of DNA strands, and then reseals the DNA backbone in two successive transesterification reactions. As illustrated in Figure 1, a key covalent topoisomerase I-DNA intermediate is formed between the tyrosine-723 residue of the enzyme and a 3'-phosphate at the break site during the transient DNA cleavage stage. The 5'-end of the broken DNA appears to be held by noncovalent interactions within the enzyme.[22,23] All the known topoisomerase I-targeting drugs damage DNA by trapping this key covalent reaction intermediate, called the “topoisomerase I cleavable complex.”[5]

Cytotoxic Mechanism of DNA Topoisomerase I-Targeting Drugs

Camptothecin and its derivatives (Figure 2) are the best characterized human topoisomerase I-targeting drugs. Camptothecin was originally isolated from the tree Camptotheca acuminata. Its broad spectrum antitumor activity in animal models, especially against colon tumors, led to clinical trials in the early 1970s. However, trials were terminated due to observations of excessive toxicity with the ring-open form of camptothecin (camptothecin, sodium salt, NSC-100880). It is now clear that the ring-open form of camptothecin is inactive against its molecular target, topoisomerase I.[24]

Among camptothecin derivatives, topotecan is positively charged and irinotecan (CPT-11[Camptosar]) is a prodrug that generates its active metabolite SN-38 intracellularly via carboxyl esteration. Both topotecan and irinotecan demonstrated efficacy in clinical trials,[24,25] and in 1997 were approved by the US Food and Drug Administration (FDA) for the treatment of recurrent colon cancer and ovarian cancer, respectively. S-phase-specific cytotoxicity,[12,13,15] selective cytotoxicity against tumorigenic over nontumorigenic cells,[26,27] and the ability to overcome MDR1-mediated drug resistance,[28,29] are features of camptothecin derivatives that may contribute to their excellent anticancer activity.

Fork Collision Model

Our current understanding of the cytotoxic mechanism of camptothecin is demonstrated by the fork collision model (Figure 3), which was proposed based on studies both in cultured cells and in cell-free extracts.[5] Upon binding of topoisomerase I to DNA, two different reaction intermediates, the “noncleavable complex” and the “cleavable complex,” are formed. In a relaxation reaction in the absence of drugs, the cleavable complex and the noncleavable complex are at equilibrium.

By inhibiting the rejoining step, topoisomerase I drugs perturb this equilibrium by trapping a reversible topoisomerase I-camptothecin-DNA ternary reaction intermediate, the topoisomerase I cleavable complex. The perturbed equilibrium can be rapidly reversed by removing drug molecules from the medium. Studies using cell synchronization techniques and specific inhibitors of DNA polymerases indicate the involvement of active DNA synthesis in the induction of the highly S-phase-specific camptothecin cytotoxicity.[30,31] It is currently hypothesized that the collision between the replication machinery and the drug-trapped topoisomerase I cleavable complex leads to eventual G2-phase cell-cycle arrest and cell death. A similar cytotoxic mechanism has been proposed for some newly identified topoisomerase I-targeting drugs, including the MGBLs Hoechst 33342 and Hoechst 33258.

Radiosensitization Mechanism of Topoisomerase I-Targeting Drugs

Understanding the mechanism of interaction between topoisomerase I-targeting drugs and ionizing radiation is a prerequisite toward successful use of their combination in cancer treatment. Controversial early studies using cultured cells and human xenografts suggested that camptothecin derivatives modulate the cytotoxic effects of ionizing radiation.[32-39] To answer key questions, such as whether camptothecin derivatives are radiosensitizers and whether DNA topoisomerase I is involved in such radiosensitization, we conducted clonogenic survival assays using cultured mammalian cells.[40] We found that drug incubation with camptothecin derivatives radiosensitized log-phased human MCF-7 breast cancer cells in a schedule-dependent manner (Table 1).[40] The radiosensitization effect was observed when the cells were exposed to drug treatment before or concurrent with radiation treatment, but not after radiation treatment (Table 1). The implication based on this observation is that camptothecin derivatives should be administered before or concurrently with radiation during chemoradiation clinical trials to optimize the radiosensitization effect.

Stereo-Specific Interaction

Camptothecin derivatives interact with DNA topoisomerase I in a stereo-specific fashion.[30] For example, assayed by the ability to induce topoisomerase I-mediated DNA cleavage, the 20(S)-stereoisomer of 10,11-methylenedioxycamptothecin is 10,000-fold more active than its 20(R)-isomer (see Figure 4A). This pair of camptothecin derivatives was used to investigate the role of DNA topoisomerase I in mediating radiosensitization. As shown in Figure 4B, only the 20(S)-10,11-methylenedioxycamptothecin radiosensitized human breast cancer MCF-7 cells. The prerequisite role of such an intact stereo-specific interaction in the induction of radiosensitization was further supported by the observation that the mutant topoisomerase I-containing DC3F cells were relatively resistant to radiosensitization.[40-42]

DNA Repair Inhibition

Some investigators have suggested DNA repair inhibition as a mechanism of radiosensitization by camptothecin derivatives.[37] If this theory is correct, a larger radiosensitizing effect should be observed in cells that are growth inhibited (G0/G1 cells) to maximize their repair function for potentially lethal damages.[37] We found that camptothecin only minimally enhanced the cytotoxic effect of radiation in plateau phase cells, which were arrested by growing to confluency, as well as in synchronized G1-phase cells obtained by mitotic shake-off technique.[40] This finding may indicate a possible therapeutic advantage of camptothecin derivatives to radiosensitize actively proliferating cancer cells selectively.

The molecular mechanism of radiosensitization of DNA topoisomerase I-targeting drugs remains to be defined. Based on available information, a plausible mechanism of radiosensitization of topoisomerase I drugs is proposed (Figure 5). It is possible that induction of radiosensitization is originally initiated by the topoisomerase I-trapped cleavable complex. This drug-stabilized cleavable complex, with a concealed single-strand DNA break, may be viewed as a “potentially sublethal” DNA damage. Interaction with as yet undefined cellular processes such as DNA replication, RNA transcription, and DNA repair may transform such “potentially sublethal” DNA damage into “sublethal” DNA damage. It is plausible that such “sublethal” DNA damage could then be converted into “lethal” DNA damage with the addition of radiation-induced DNA damage.

Clinical Chemoradiation Trials of Camptothecin Derivatives

All of the topoisomerase I-targeting drugs currently in clinical development are camptothecin derivatives. Among them, irinotecan, topotecan, and 9-aminocamptothecin are the most extensively studied.[25,26] A wide spectrum of clinical antitumor activity, including activity against gastrointestinal tract cancer, ovarian cancer, small-cell lung cancer, non–small-cell lung cancer, and malignant lymphomas has been observed with camptothecin derivatives.[25,26] Based on clinical success as systemic therapy, chemoradiation trials of irinotecan and topotecan for a variety of solid tumors are currently ongoing.

Phase I/II Trials

Table 2 shows some clinical phase I/II chemoradiation trials of irinotecan (CPT-11) for patients with locally advanced non–small-cell lung cancer.[43-46] In general, impressive objective response rates ranging from 60% to 80% have been achieved in patients treated with various chemoradiation combinations with irinotecan and carboplatin. The incidence of grade 3 or greater treatment-related toxicities (ie, fever, neutropenia, thrombocytopenia, pneumonitis, and esophagitis) was low (Table 2). The regimen of concurrent radiation with weekly carboplatin and irinotecan will most likely be adopted by the Radiation Therapy Oncology Group (RTOG) as one treatment arm in a new randomized phase II trial in patients with locally advanced non–small-cell lung cancer. The efficacy of irinotecan, either used as second-line treatment for 5-FU-refractory tumors or as first-line treatment for colorectal cancer, has also been well established by clinical trials.[47] Clinical trials using combination therapy with irinotecan and radiation for locally advanced colorectal cancer appear to be the next logical step in improving treatment outcomes in this group of patients.

The clinical use of topotecan in the chemoradiation setting is currently being explored in non–small-cell lung cancer and tumors of the central nervous system. A combination of cranial irradiation and topotecan is being evaluated by the RTOG and the Children’s Cancer Group (CCG) in patients with glioblastoma and pontine gliomas, respectively.

Conclusions and Future Directions

In conclusion, an intact stereo-specific interaction between drug molecule and topoisomerase I is a prerequisite step for the induction of DNA topoisomerase I-mediated, schedule-dependent radiosensitization in mammalian cells. Future studies are needed to increase our understanding of the mechanisms underlying radiosensitization by camptothecin derivatives. As yet unresolved issues include mechanisms of schedule dependence, the role of cell-cycle distribution, the influence of hypoxia, as well as the role of DNA replication and repair. It will also be interesting to test the potential radiosensitizing effects of other new topoisomerase I-targeting drugs such as the DNA MGBLs.[5,40]

An understanding of the molecular radiosensitization mechanism induced by topoisomerase I-targeting antitumor drugs may also facilitate the development of more effective radiosensitizers for cancer treatment. Only when the detailed mechanisms of interaction between drugs and radiation have been established can efficacious, novel chemoradiation regimens be accurately designed and tailored against different kinds of cancer. Chemoradiation using topoisomerase I-targeting drugs is currently being investigated and may prove to be an effective therapy against several kinds of human solid tumors. Recent advances in the field of DNA topoisomerase I provide a unique opportunity to translate basic science research into improvements in cancer treatment.


1. Hellman S: Principles of cancer management: Radiation therapy, in DeVita VT Jr., Hellman S, Rosenberg SA (eds): Cancer: Principles and Practice of Oncology, 5th ed, pp 307-332. Philadelphia, JB Lippincott Company, 1997.

2. Rotman M, Aziz H, Wasserman TH: Chemotherapy and irradiation, in Perez CA, Brady LW (eds): Principles and Practice of Radiation Oncology, 3rd ed, pp 705-722. Philadelphia, JB Lippincott Company, 1998.

3. Cook JA, Mitchell JB: Radiation and chemotherapy cytotoxicity: Mechanisms of resistance, in John MJ, Flam MS, Legha SS, et al (eds): Chemoradiation: An Integrated Approach to Cancer Treatment, pp 159-180. Philadelphia, JB Lippincott Company, 1995.

4. McGinn CJ, Shewach, DS, Lawrence TS: Radiosensitizing nucleotides. J Natl Cancer Inst 88:1193-1203, 1996.

5. Chen AY, Liu LF: DNA topoisomerases: Essential enzymes and lethal targets. Annu Rev Pharmacol Toxicol 34:191-218, 1994.

6. Hsiang YH, Hertzberg R, Hecht S, et al: Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 260:14873-14878, 1985.

7. Hsiang YH, Liu LF: Identification of mammalian DNA topoisomerase I as an intracellular target of the anticancer drug camptothecin. Cancer Res 48:1722-1726, 1988.

8. Andoh T, Ishii K, Suzuki Y, et al: Characterization of a mammalian mutant with a camptothecin-resistant DNA topoisomerase I. Proc Natl Acad Sci USA 84:5565-5569, 1987.

9. Nitiss J, Wang JC: DNA topoisomerase-targeting antitumor drugs can be studied in yeast. Proc Natl Acad Sci USA 85:7501-7505, 1988.

10. Chen AY, Yu C, Gatto B, et al: DNA minor groove-binding ligands: A different class of mammalian DNA topoisomerase I inhibitors. Proc Natl Acad Sci USA 90:8131-8135, 1993.

11. Chen AY, Yu C, Bodley A, et al: A new mammalian DNA topoisomerase I poison Hoechst 33342: Cytotoxicity and drug resistance in human cell cultures. Cancer Res 53:1332-1337, 1993.

12. Hsiang YH, Lihou MG, Liu LF: Arrest of replication forks by drug- stabilized topoisomerase I-DNA cleavable complexes as a mechanism of cell killing by camptothecin. Cancer Res 49:5077-5082, 1989.

13. Holm C, Covey JM, Kerrigan D, et al: Differential requirement of DNA replication for the cytotoxicity of DNA topoisomerase I and II inhibitors in Chinese hamster DC3F cells. Cancer Res 49:6365-6368, 1989.

14. Zhang H, D’Arpa P, Liu LF: A model for tumor cell killing by topoisomerase poisons. Cancer Cells 2:23-27, 1990.

15. D’Arpa P, Beardmore C, Liu LF: Involvement of nucleic acid synthesis in cell killing mechanisms of topoisomerase poisons. Cancer Res 50:6919-6924, 1990.

16. Giovanella BC, Stehlin JS, Wall ME, et al: DNA topoisomerase I-targeted chemotherapy of human colon cancer in xenografts. Science 246:1046-1048, 1989.

17. Potmesil M, Hsiang YH, Liu LF, et al: Resistance of human leukemic and normal lymphocytes to drug-induced DNA cleavage and low levels of DNA topoisomerase II. Cancer Res 48:3537-3543,1988.

18. Hwang JL, Shyy SH, Chen AY, et al: Studies of topoisomerase-specific antitumor drugs in human lymphocytes using rabbit antisera against recombinant human topoisomerase II polypeptide. Cancer Res 49:958-962, 1989.

19. Gellert M: DNA topoisomerases. Annu Rev Biochem 50:879-910, 1981.

20. Wang JC: DNA topoisomerases. Annu Rev Biochem 54:665-697, 1985.

21. Wang JC: DNA topoisomerases. Annu Rev Biochem 65:635-692, 1996.

22. Duguet M, Lavenot C, Harper F, et al: DNA topoisomerases from rat liver: Physiological variations. Nucleic Acids Res 11:1059-1075, 1983.

23. Liu LF, Miller KG: Eukaryotic DNA topoisomerases: two forms of type I DNA topoisomerases from HeLa cell nuclei. Proc Natl Acad Sci USA 78:3487-3491, 1981.

24. Burke TG: Chemistry of the camptothecins in the blood stream: Drug stabilization and optimization of activity, in Pantazis P, Giovanella BC, Rothenberg ML (eds): The Camptothecins From Discovery to the Patient, pp 29-31. New York, New York Academy of Sciences, 1996.

25. Clinical status and future directions of irinotecan. Proceedings of a symposium. Houston, Texas, USA. January 21-23, 1998. Oncology 12(suppl 6):1-128, 1998.

26. Pantazis P, Early JA, Kozielski AJ, et al: Regression of human breast carcinoma tumors in immunodeficient mice treated with 9-nitrocamptothecin: Differential response of nontumorigenic and tumorigenic human breast cells in vitro. Cancer Res 53:1577-1582, 1993.

27. Pantazis P, Kozielski AJ, Mendoza JT, et al: Camptothecin derivatives induce regression of human ovarian carcinomas grown in nude mice and distinguish between nontumorigenic and tumorigenic cells in vitro. Int J Cancer 53:863-871, 1993.

28. Chen AY, Yu C, Pomesil M, et al: Camptothecin overcomes MDR1-mediated resistance in human KB carcinoma cells. Cancer Res 51:6039-6044, 1991.

29. Chen AY, Liu LF: Design of topoisomerase inhibitors to overcome MDR1-mediated drug resistance in human cancers, in Liu LF (ed): DNA Topoisomerases, pp 245-256. San Diego, Kluwer Academic Press, 1994.

30. Jaxel C, Kohn KW, Wani MC, et al: Structure-activity study of the actions of camptothecin derivatives on mammalian topoisomerase I: Evidence for a specific receptor site and a relation to antitumor activity. Cancer Res 49:1465-1469, 1989.

31. Hsiang YH, Liu LF, Wall ME, et al: DNA topoisomerase I-mediated DNA cleavage and cytotoxicity of camptothecin analogues. Cancer Res 49:4385-4389, 1989.

32. Boothman DA, Trask DK, Pardee AB: Inhibition of potentially lethal DNA damage repair in human tumor cells by beta-Lapachone, an activator of topoisomerase I. Cancer Res 49:605-612, 1989.

33. Mattern MR, Hofmann GA, McCabe FL, et al: Synergistic cell killing by ionizing radiation and topoisomerase I inhibitor topotecan (SK&F 104864). Cancer Res 51:5813-5816, 1991.

34. Kim JH, Kim SH, Kolozsvary A, et al: Potentiation of radiation response in human carcinoma cells in vitro and murine fibrosarcoma in vivo by topotecan, an inhibitor of DNA topoisomerase I. Int J Radiat Oncol Biol Phys 22:515-518, 1992.

35. Boothman DA, Wang M, Schea, RA, et al: Posttreatment exposure to camptothecin enhances the lethal effects of x-rays on radioresistant human malignant melanoma cells. Int J Radiat Oncol Biol Phys 24:939-948, 1992.

36. Roffler SR, Chan J, Yeh MY: Potentiation of radioimmunotherapy by inhibition of topoisomerase I. Cancer Res 54:1276-1285, 1994.

37. Boothman DA, Fukunaga N, Wang M: Down-regulation of topoisomerase I in mammalian cells following ionizing radiation. Cancer Res 54:4618-4626, 1994.

38. Falk SJ, Smith PJ: DNA damaging and cell cycle effects of the topoisomerase I poison camptothecin in irradiated human cells. Int J Radiat Biol 61:749-757, 1992.

39. Hennequin C, Giocanti N, Balosso J, et al: Interaction of ionizing radiation with the topoisomerase I poison camptothecin in growing V-79 and HeLa cells. Cancer Res 54:1720-1728, 1994.

40. Chen AY, Okunieff P, Pommier Y, et al: Mammalian DNA topoisomerase I mediates the enhancement of radiation cytotoxicity by camptothecin derivatives. Cancer Res 57:1529-36, 1997.

41. Tanizawa A, Pommier Y: Topoisomerase I alteration in a camptothecin-resistant cell line derived from Chinese hamster DC3F cells in culture. Cancer Res 52:1848-1854, 1992.

42. Tanizawa A, Beitrand R, Kohlhagen G, et al: Cloning of Chinese hamster DNA topoisomerase I cDNA and identification of a single point mutation responsible for camptothecin resistance. J Biol Chem 268:25463-25468, 1993.

43. Kudoh S, Kurihara N, Okishio K, et al: A phase I-II study of weekly irinotecan (CPT-11) and simultaneous thoracic radiotherapy (TRT) for unresectable locally advanced non–small-cell lung cancer (NSCLC) (abstract 1102) Proc Am Soc Clin Oncol 15:372, 1996.

44. Saka H, Shimokata K, Yoshida S, et al: Irinotecan (CPT-11) and concurrent radiotherapy in locally advanced non–small-cell lung cancer (NSCLC): A phase II study of Japan Clinical Oncology Group (JCOG9504) (abstract 1607) Proc Am Soc Clin Oncol 16:447a, 1997.

45. Okamoto H, Nagatomo A, Kunitoh H, et al: A combination phase I-II study of carboplatin/irinotecan plus G-CSF with the Calvert equation in patients with advanced non–small-cell lung cancer (NSCLC) (abstract 1150) Proc Am Soc Clin Oncol 14:373, 1995.

46. Nakagawa K, Yamamoto N, Kudoh S, et al: Irinotecan (CPT-11) and carboplatin (CBDCA) with concurrent thoracic radiotherapy (TRT) for unresectable, stage III non–small-cell lung cancer (NSCLC)—Preliminary results (abstract 1909). Proc Am Soc Clin Oncol 17:496a, 1998.

47. Rothenberg ML, Blanke CD: Topoisomerase I inhibitors in the treatment of colorectal cancer. Oncology 1999, in press.

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