hr. J. Radiarion Oncology Rid. Phys.. Vol. 22. pp. 515-518 Pnnted in the U.S.A. All rights reserved


036%3016192 $5.00 + 00 6 1992 Pergamon Press plc

??Session B: Biochemical Modification of Therapeutic Response






Henry Ford Hospital, Department of Radiation Oncology, 2799 W. Grand Boulevard, Detroit, MI 48202 DNA topoisomerase I, a nuclear enzyme important for solving topologic problems arising during DNA replication, has been identified as a principal target of a plant alkaloid, 20(s)-camptothecin. In view of the profound biochemical effects of camptothecin and its analogues on DNA replication and the differential cytotoxic effects on human tumors in xenografts, experiments were performed to determine whether topotecan, a camptothecin analogue, would potentiate the radiation effects on human carcinoma cells in culture and murine fibrosarcoma in mice. Cell culture studies showed that a dose dependent reduction in cell survival was obtained with a 4 hr exposure of the drug following irradiation of cells. No enhancement of cell killing was seen when cells were treated with the drug before irradiation. Preliinary in vivo tumor studies showed a significant radiosensitizhtg effect of topotecan that was both drug dose (20 mg/kg) and time sequence (4 hr before irradiation) dependent. There was no enhanced skin reaction following the combined treatments. Topotecan, Radiosensitization, HeLa cells, Meth-A tumor. METHODS



Experiments were carried out with HeLa S-3 cells grown in Eagle’s minimal essential medium supplemented with 10% fetal calf serum. Details of the cell culture procedures, including the maintenance, the trypsinization, and the test for contamination of cultures with Mycloplasma, have been described elsewhere (7). No antifungal agent was used throughout the study. Cell survival was assayed by the colony-forming ability of plated single cells to obtain quantitative dose-survival curves. Topotecan was obtained through the generosity of Dr. R. Johnson of the Smith Kline and French Laboratories, PA. Methylcholanthrene induced fibrosarcomas (Meth-A tumor) grown in isogenic male BALB/c mice were used in the present study (8). Six- to eight-week-old BALB/c mice were inoculated with single cell suspensions of Meth-A cells obtained from donor mice, in which the tumor was maintained in the ascitic form. For tumor transplantation, cells were harvested in the ascitic form, washed in phosphate-buffered saline, centrifuged, and re-suspended in heparinized minimum essential medium for inoculation. lo6 viable tumor cells were transferred intramuscularly to the distal thigh region. Irradiation was administered to anesthetized mice (ketamine HCl, 125 mg/kg) post-transplantation. A @?o Ther-

DNA topoisomerase I, a nuclear enzyme important for solving topological problems arising during DNA replication and for other cellular functions, has been identified as a principal target of a plant alkaloid, 20(s)-camptothecin (10). High levels of the enzyme in several types of human cancer and low levels in corresponding normal tissues may represent a therapeutic advantage in cancer treatment with biologically active analogues of camptothecin (3). More recently, topoisomerase I has been implicated in the DNA repair processes for x-ray induced damage (1). Topotecan is a semisynthetic analogue of the alkaloid camptothecin. Like camptothecin, the drug is a specific inhibitor of topoisomerase I (6). The inhibition of this enzyme results in lethal damage during the course of DNA replication. Because of a high degree of activity in a broad spectrum of animal tumor models, topotecan is currently undergoing Phase I clinical trials. In view of the profound biochemical effects of camptothecin and its analogues on DNA replication and the differential cytotoxic effects on human tumors in xenografts, experiments were performed to determine whether topotecan, a camptothecin analogue, would potentiate the cytotoxic effects of radiation on human carcinoma cells in culture and murine fibrosarcoma in BALB/c mice.

Accepted for publication 3 July 1991.

Reprint requests to: Jae Ho Kim, M.D., Ph.D., FACR. 515

I. J. Radiation Oncology 0 Biology 0 Physics

Volume 22, Number 3, 1992 Preirradiation

101 0


I 4


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Radiation Dose (Gy) I 4

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Fig. 1. Effects of topotecan on the cytotoxicity of HeLa cells as a function of drug concentration and of exposure time. Cells exposed to the drug for 4 hr at concentrations ranging from 1 to 8 p.M (top). Cells exposed to the drug (4 PM) for varying periods of time (bottom). Error bars indicate SEM.

atron was used at a dose rate of 2 Gy/min. The experiments were initiated when the average diameter of the tumors was 8 mm. Follow-up evaluation after treatment included measurement of tumor size three times per week. Local tumor control was defined as a non-palpable tumor at 60 days post-treatment.

Fig. 2. Cell survival curves as a function of radiation dose. Cells were either exposed to the drug before irradiation (pre-) for 4 hr or after irradiation (post-) for 4 hr. 0 control; A 2.0 FM; n 4.0 FM; ??8.0 FM. Error bars indicate SEM.

PM in non-irradiated nificantly (Fig. 1).

cells did not increase cell killing sig-

The potentiation of radiation response on Meth-A tumor by topotecan Figure 4 shows the tumor growth delay as a function of time after radiation alone or in combination with a single

RESULTS Cytotoxicity of topotecan to HeLa cells Figure 1 shows the effects of topotecan on the viability of HeLa cells as a function of drug concentration and of exposure time. The shape of the cell survival curve after exposure to the drug exhibited a bi-phasic exponential curve. Four-hour exposure to the drug produced limited cytotoxicity with 3040% survivals at drug concentrations ranging from 1 to 8 FM (Fig. 1, Top). Time dependence of drug-induced cytotoxicity is shown in the bottom portion of Figure 1. A short 1-hr exposure to the drug (4 ~_LM)produced more than a 50% cell survival. Prolonged exposure of up to 6 hr produced a gradual reduction in the viability of cells. Effect of topotecan on irradiated HeLu cells Figure 2 shows cell survival curves treated with varying concentrations of topotecan for 4 hr, either before or immediately after irradiation. It is evident that cells exposed to topotecan immediately after irradiation showed drug dose dependent radiosensitization. On the other hand,‘cells exposed to the drug before irradiation showed no enhancement of the radiation effects, even at the highest concentration, 8 PM. The drug concentration dependence of radiosensitization of cells after radiation is shown in Figure 3. Note that increasing drug concentrations above 1

Drug alone 0




Fig. 3. Percent cell survival as a function of the drug concentration. Cells were exposed to varying concentrations of the drug (4 hr) before or after a single dose of irradiation (4 Gy). Error bars

indicate SEM.




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Drug -> 2 hrs -> RT


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10 Days



0 J. H. KIM et al.



Fig. 4. Tumor growth delay of Meth-A fibrosarcoma after a single dose of radiation (25 Gy). A single dose of topotecan (20 mg/kg) was administered to mice at 2 hr and 4 hr before irradiation. The numbers in parentheses refer to recurrences. Error bars indicate SEM.

dose of topotecan applied before or after radiation. There was no significant growth delay observed with the drug alone (20 mg/kg). The enhanced radiosensitizing effect of the drug was obtained at two hours and four hours before radiation. The drug was administered several hours before irradiation, in order to achieve an adequate drug concentration within the tumor tissue at the time of irradiation. The administration of the drug 2 hr after irradiation did not produce any significant potentiation at the concentration used. The overall local tumor control rate following the combined treatment was 75% when the drug was applied before irradiation, whereas radiation alone (25 Gy) produced 0%. This factor suggests that the drug effects may be even more evident under conditions of fractionated radiation treatments, that is, with low doses per fraction.

DISCUSSION The data presented herein demonstrate that radiation, in combination with topotecan, significantly increases the radiation-induced cell lethality of HeLa cells in culture and of Meth-A fibrosarcoma in BALB/c mice. The degree of radiosensitization by the drug appears dependent on the drug concentration and the time and sequence of drug administration with respect to radiation (Figs. 3 and 4). The abrupt loss of cell viability following exposure of asynchronously growing cells to topotecan suggests that a fraction of the cell population in the particular phase during the cell cycle might be more sensitive to the drug (Fig. 1). There exists some evidence that the most sensitive cells to the drug during the cell cycle would be cells in the S and G, phases (2, 5). It has also been shown that the cytotoxicity of camptothecin could be abrogated by exposure of cells to aphidocolin, an inhibitor of DNA synthesis (5). Although the radiation response of drug treated cells might


represent the response of selected cell populations by the drug toxicity, the significant enhancement of the radiation response of cells to the drug only after irradiation, and not before irradiation, suggests that the drug inhibits the postradiation repair process. Experiments with synchronized cells would permit an easier analysis of the combined effect of the drug and radiation. The present cell culture data with topotecan are in agreement with the concept that DNA topoisomerase I is involved with the post-radiation repair process (1). Camptothecin, an inhibitor of topoisomerase I, has been shown to be an inhibitor of potentially lethal damage repair (PLDR) in a cell culture system (1). Although topoisomerase I is intimately involved in DNA replication as it relieves the torsional strain introduced ahead of the moving replication of DNA, topoisomerase I activity is not directly linked to the proliferation rate, and similar enzyme activities are detected in slowly proliferating or quiescent cells (10). Indeed, topoisomerase I is present in many human tumors that have the potential of slow proliferation (3). Therefore, the inhibition of topoisomerase I will increase radiation effects regardless of the state of growth of the tumor cells. The degree and pattern of sensitizing efficacy of radiation by topotecan in Meth-A tumors are quite similar to our previous studies obtained with other inhibitors of PLDR, including fludarabine phosphate, 6-thioguanine, and lonidamine (4, 8, 9). In the present study, more than a 75% cure rate can be obtained with a single injection of topotecan (20 mg/kg) after a single dose of radiation (25 Gy). The LD,, of a single dose of intraperitoneal administration of topotecan in BALBlc mice is 30 mg/kg. Although the drug has been shown to be effective in a number of solid tumor models (e.g., Lewis lung carcinoma, colon carcinomas 38 and 5 1, and B-16 melanoma), the administration of topotecan by itself at a concentration of 20 mg/kg did not produce any detectable growth delay. Several inhibitors of PLDR have been tested in in viva tumor systems as potential radiosensitizers. Purine nucleoside analogues were found to enhance tumor control rates (11, 13). The compounds tested, however, were reported to be too toxic for human use. B-Ara-A and its analogue, fludarabine phosphate, are being tested in humans (7, 13). Unlike purine nucleoside analogues, inhibitors of topoisomerase are unique in that they do not cause cytotoxicity by depleting the product of their target enzymes, but by producing DNA damage by interfering with topoisomerase function. Therefore, the drug activity is directly proportional to target enzyme levels, rather than to the converse which is the case for many cytotoxic enzyme inhibitors. Although the present study was not aimed at designing the optimum dose fractionation scheme, several radiobiological and pharmacokinetic factors should be further investigated prior to future clinical trials. These data should provide useful information as to either the rate or frequency of drug administration, since the kinetics of PLDR repair in human tumors may be as long as 24 hours after


I. J. Radiation Oncology ??Biology 0 Physics

irradiation (12, 14). Another important consideration for topotecan use in combination with radiation is its effects on normal tissues. Our preliminary studies on the skin reaction of mice exposed to the combined treatments, when

Volume 22, Number 3, 1992

compared to the radiation alone, showed no disproportionately enhanced reaction. However, more detailed studies are needed on the other dose-limiting normal tissues under similar conditions.

REFERENCES 1. Boothman, D. A.; Trask, D.K.; Pardee, A. B. Inhibition of potentially lethal DNA damage repair in human tumor cells by B-lapachone, an activator of topoisomerase I. Cancer Res. 49605612; 1989. 2. Del Bino, G.; Skierski, J. S.; Darzynkiewicz, Z. Diverse effects of camptothecin, an inhibitor of topoisomerase I, on the cell cycle of lymphocytic and myelogeneous leukemic cells. Cancer Res. 505746-5750; 1990. 3. Giovanella, B. C.; Stehlin, J. S.; Wall, M. E.; Wani, M. C.; Nicholas, A. W.; Liu, L. F.; Silber, R.; Potmesil, M. DNA topoisomerase I-targeted chemotherapy of human colon cancer in xenografts. Science 246: 1046-1048; 1989. 4. Hahn, G. M.; Van Kersen, I.; Silvestrini, B. Inhibition of the recovery from potentially lethal damage by lonidamine. Br. J. Cancer 50:657-660; 1984. 5. Holm, C.; Covey, J. M.; Kerrigan, D.; Pommier, Y. 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. 6. Hsiang, Y. W.; Liu, L. F.; Wall, M. E.; Wani, M. C.; Nicholas, A. W.; Manikumar, G.; Kirschenbaum, S.; Silber, R.; Potmesil, M. DNA topoisomerase I-mediated DNA cleavage and cytotoxicity of camptothecin analogues. Cancer Res. 49:43854389; 1989. 7. Kim, J. H.; Alfieri, A. A.; Kim, S. H.; Fuks, Z. The potentiation of radiation response on murine tumor by fludarabine


Cancer Lett. 31:69-76;


8. Kim, J. H.; Alfieri, A. A.; Kim, S. H.; Young, C. W. Po9.

10. Il.




tentiation of radiation effects on two murine tumors by lonidamine. Cancer Res. 46:1120-l 123; 1986. Kim, J. H.; Alfieri, A. A.; Kim, S. H.; Hong, S. S. Radiosensitization of two murine fibrosarcomas with 6-thioguanine. Int. J. Radiat. Oncol. Biol. Phys. 18:583-586; 1990. Liu, L. F. DNA topoisomerase poisons as anti-tumor drugs. Ann. Rev. Biochem. 58:351-375; 1989. Nakatsugawa, S.; Sugahara, T. Inhibition of x-ray induced potentially lethal damage (PLD) repair by cordycepin (3’deoxyadenosine) enhancement of its action by 2’-deoxycoformycin in Chinese hamster hai cells in the stationary phase in vitro. Radiat. Res. 84:265-275; 1980. Phillips, R. A.; Tolmach, L. J. Repair of potentially lethal damage in x-irradiated HeLa cells. Radiat. Res. 29:414-432; 1966. Sougawa, M.; Akagi, K.; Murata, T.; Kawasaki, S.; Sawada, S.; Yoshii, G.; Tanaka, Y. Enhancement of radiation effects by acyclovir. Int. J. Radiat. Oncol. Biol. Phys. 12:1537-1540; 1986. Weichselbaum, R. J.; Little, J. B. The differential response of human tumors to fractionated radiation may be due to a post-radiation repair process. Br. J. Cancer 46:532-537; 1982.

Potentiation of radiation response in human carcinoma cells in vitro and murine fibrosarcoma in vivo by topotecan, an inhibitor of DNA topoisomerase I.

DNA topoisomerase I, a nuclear enzyme important for solving topologic problems arising during DNA replication, has been identified as a principal targ...
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