Chromosome damage induced by DNA topoisomerase II inhibitors combined with g-radiation in vitro

Chromosome damage induced by DNA topoisomerase II inhibitors combined with g-radiation in vitro

Maria Cristina P. Araújo^1,2, Francisca da Luz Dias¹, Andréa O. Cecchi¹, Lusânia M.G. Antunes¹ and Catarina S. Takahashi^1,3

¹Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900, 14049-900 Ribeirão Preto, SP, Brasil. E-mail: [e-mail] Send correspondence to M.C.P.A.

²Departamento de Genética, Universidade Federal de Pernambuco and ³Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Brasil.

SUMMARY

Combined radiation and antineoplastic drug treatment have important applications in cancer therapy. In the present work, an evaluation was made of two known topoisomerase II inhibitors, doxorubicin (DXR) and mitoxantrone (MXN), with g-radiation. The effects of DXR or MXN on g-radiation-induced chromosome aberrations in Chinese hamster ovary (CHO) cells were analyzed. Two concentrations of each drug, 0.5 and 1.0 µg/ml DXR, and 0.02 and 0.04 µg/ml MXN, were applied in combination with two doses of g-radiation (20 and 40 cGy). A significant potentiating effect on chromosomal aberrations was observed in CHO cells exposed to 1.0 µg/ml DXR plus 40 cGy. In the other tests, the combination of g-radiation with DXR or MXN gave approximately additive effects. Reduced mitotic indices reflected higher toxicity of the drugs when combined with radiation.

INTRODUCTION

Topoisomerases are enzymes involved in the maintenance of DNA structure (Ferguson and Baguley, 1994). Topoisomerase II is an essential enzyme in eukaryotic cells and is thought to play an important role in DNA replication (Mattern and Painter, 1979), transcription (Rowe et al., 1986) and cell division (Holm et al., 1985). This enzyme catalyzes the breaking and rejoining of both DNA strands (Palitti et al., 1994). In general, DNA topoisomerase II inhibitors result in the formation of a DNA-drug-enzyme complex that may lead to induction of chromosomal mutations in mammalian cells (De Marini and Lawrence, 1992). Certain antitumor drugs, including anthracyclines, anthracenediones, ellipticines and acridines, have been found to stabilize the cleavable complex (Chen et al., 1984; Tewey et al., 1984). Another major effect of topoisomerase II-directed agents on cycling cells is the induction of programmed cell death or apoptosis (Ferguson and Baguley, 1994).

Many of the clinical topoisomerase II inhibitors, such as doxorubicin and mitoxantrone, are clinically important antitumor agents, which intercalate into DNA (Liu, 1989; Lown, 1993). Both mutagenicity and clastogenicity are related directly to cytotoxicity and topoisomerase II inhibitor activity (Ferguson et al., 1989).

The anthracycline antibiotic doxorubicin (DXR) is a chemotherapeutic agent with strong activity against a wide range of human malignant neoplasms (Young et al., 1981; O'Shaughnessy et al., 1994; Sledge Jr. et al., 1995). The genotoxicity of DXR may be attributed to its ability to stabilize cleavable topoisomerase II-DNA complexes (Ross et al., 1979; Zunino et al., 1980; Goldenberg et al., 1986), intercalate DNA (Cummings et al., 1992) and generate reactive oxygen species (Myers et al., 1977; Keizer et al., 1990).

The antineoplastic agent mitoxantrone (MXN) is an anthracenedione with significant cytostatic activity against a number of experimental tumors and human malignancies (Alberts et al., 1988). Despite intensive investigation, the mechanisms of action of this drug have not been fully defined. Intercalation, electrostatic interaction with DNA and modulation of topoisomerase II are considered to be responsible for its cytotoxicity (Blanz et al., 1991). It has also been demonstrated that MXN is clastogenic in in vitro (Medeiros and Takahashi, 1994) and in vivo (Cecchi et al., 1996) experiments performed on mammalian cells.

A primary type of DNA damage induced by topoisomerase II inhibitors is DNA-strand breakage, which is also a major type of DNA damage induced by ionizing radiation and reactive oxygen species. Ionizing radiation is cytotoxic to cells as a direct consequence of DNA damage (Painter and Young, 1980). It induces chromosomal aberrations, mutations and neoplastic transformation (Obe et al., 1992). Among the different types of DNA damage induced by ionizing radiation, double strand breaks constitute the most important lesions in terms of provoking cell death (Iliakis, 1991).

It is clear that topoisomerase II inhibitors can induce chromosomal aberrations in mammalian cells (De Marini and Lawrence, 1992), but the effect of g-radiation on this type of damage is not well understood. This study was undertaken to evaluate the effects of g-radiation on DXR- or MXN-induced damage in Chinese hamster ovary (CHO) cells by analyzing chromosomal aberrations and mitotic indices.

MATERIAL AND METHODS

Agents

Doxorubicin (Adriblastina^®) was donated by Farmitalia Carlo Erba, Brazil. Mitoxantrone (Novantrone^â) was obtained from the Hospital das Clínicas, Faculdade de Medicina de Ribeirão Preto (FMRP), Universidade de São Paulo (USP), SP, Brazil. The chemicals were dissolved with distilled water just before use. Cells were acutely irradiated at a dose rate of 84.386 cGy/min using a Siemens Gammatron S-80 source (Hospital das Clínicas, FMRP - USP).

Chinese hamster ovary cells

Chinese hamster ovary cells (CHO-9 line) were kindly supplied by Prof. A. T. Natarajan (University of Leiden, The Netherlands). Cells were maintained as monolayers growing at 37°C in 25-cm² flasks (Corning) containing HAM-F10 plus DMEM (Dulbecco's modified Eagle's medium; Sigma) medium (1:1 ratio), supplemented with 10% fetal calf serum (Cultilab), antibiotics (0.1 mg/ml streptomycin and 0.06 mg/ml penicillin) and 2.38 mg/ml Hepes (Sigma). Cells were subcultured two or three times a week, washed in phosphate buffered saline (PBS) and treated with ATV (0.2% trypsin and 0.02% versene - Instituto Adolfo Lutz) for removal from the inner surface of the culture flask.

Exponentially growing CHO cells, with a doubling time of 12-14 h (Preston et al., 1981), were seeded (1 x 10⁶ cells/flask) and then exposed to DXR (0.5 or 1.0 µg/ml) or MXN (0.02 or 0.04 mg/ml) for 60 min at 37°C. After treatment, cells were washed twice with PBS, fed with fresh medium, immediately exposed to g-radiation (20 or 40 cGy), and harvested three hours later. According to Preston et al. (1981), cells fixed three hours after the beginning of treatment are probably in the G₂ phase of the cell cycle. For control, cells were exposed to DXR, MXN and g-radiation, separately. Untreated controls were handled identically, with comparable medium changes. Colcemide (100 µl at a concentration of 5 µg/ml) was added to the culture medium two hours before harvesting. Each experiment was repeated three times.

Cells were harvested by the method of Moorhead et al. (1960) with some modifications (1% sodium citrate hypotonic solution, methanol/acetic acid 3:1 fixative). The air-dried chromosome preparations were stained with 3% Giemsa diluted in phosphate buffer. Only well-spread metaphases with 21 ± 1 chromosomes were analyzed. Three hundred metaphases were analyzed per treatment in order to determine the frequencies of chromosomal aberrations (blind test). The mitotic index was obtained by counting the number of mitotic cells out of 6000 cells analyzed per treatment.

Statistical analysis

Data concerning number of abnormal metaphases and total number of chromosomal aberrations, including or excluding gaps, and mitotic index were analyzed statistically by analysis of variance, with calculations of the F statistic and its respective P value. In all cases in which P < 0.05, the mean values of each treatment were compared by the Tukey test, with calculation of the minimum significant difference for P = 0.05.

RESULTS

Clastogenesis induced by g-radiation in CHO cells increased in a dose-dependent manner (Tables I and ^II), and both abnormal metaphases (AM) and total number of chromosome aberrations (TCA) significantly increased compared to the untreated control (P < 0.05). Chromatid breaks were the most frequent type of damage observed, followed by chromatid gaps. The mitotic indices (MI) observed in irradiated cultures were not significantly different from those found in untreated controls.

DXR produced a significant increase in AM and TCA frequencies when compared to the untreated control (Table I, P < 0.05). This effect was even higher when 1.0 mg/ml DXR was used. Among the chromosome aberrations detected in treatments with DXR in combination or not with g-radiation, chromatid breaks were the most frequent, followed by chromatid gaps. Other alterations such as isochromatid breaks, rings, dicentrics, quadriradials and triradials were observed at lower frequencies.

All groups treated with DXR plus g-radiation showed not significantly increased AM and TCA frequencies when compared to the sum of the respective controls, with the effect being close to additive. However, only when 1.0 mg/ml of DXR was combined with 40 cGy did we note a potentiating effect in the total number of abnormal metaphases which was statistically significant (P < 0.05; Table I). Cultures treated with DXR showed a lower MI than the untreated controls and this decrease was statistically significant to both DXR 1.0 µg/ml alone and DXR 1.0 µg/ml in association with 40 cGy (P < 0.05; Table I).

The results obtained with MXN combined or not with g-radiation were similar to those obtained with DXR combined or not with g-radiation. CHO cells treated with MXN produced a significantly higher frequency of AM and TCA than the untreated controls (^{Table II}, P < 0.05). Chromatid breaks and chromatid gaps were the most common type of chromosomal damage induced by MXN combined or not with g-radiation. In all MXN plus g-radiation combinations tested, there was an increase in the frequency of aberrations compared to groups treated with MXN or g-radiation alone, but this effect was not statistically significant and remained close to the additive effect. There were significant differences in MI values between groups treated with MXN and untreated controls (P < 0.05). However, cytotoxicity in groups treated with MXN plus g-radiation was greater than that observed in groups treated with MXN or g-radiation separately (^{Table II}).

Besides the doses used (20 and 40 cGy), it was also tested 60 cGy of g-radiation combined with either DXR or MXN concentrations. However, this dose of g-radiation associated with DXR or MXN was strongly cytotoxic and these data will not be presented.

DISCUSSION

Topoisomerase II is elevated in rapidly proliferating cells; it increases during S phase and reaches its peak during the G2/M phase of the cell cycle (Anderson and Berger, 1994). It is well known from radiobiological principles that G2/M is the most radiosensitive phase of the cell cycle (Sinclair, 1968). As topoisomerase II inhibitors also arrest cells in G2/M (Del Bino et al., 1992), all experiments were undertaken in the G2 phase of the cell cycle.

Since radiation exposure has no direct effect on topoisomerase II activity (Walters et al., 1989), we investigated the influence of DXR or MXN, two known cytotoxic drugs and topoisomerase II inhibitors, on the radiation response of CHO cells by analysis of chromosomal aberrations and mitotic index. Inhibitors of DNA topoisomerases appear to be a promising tool to be used to understand the mechanisms of chromosomal damage and their kinetics (Palitti et al., 1994).

Concentrations and time of exposure to either DXR or MXN were chosen based on their cytotoxicity, estimated by reduction in mitotic indices, observed in experiments in which each drug was tested separately. CHO cells were exposed during 1 h to low concentrations of either DXR or MXN, resulting in clastogenic and cytotoxic effects. Short exposure to low concentrations of other DNA topoisomerase inhibitors has also produced cytotoxic effects (Pommier et al., 1987; Noviello et al., 1994).

We observed that the frequency of chromosome aberrations and altered cells in cultures treated with DXR or MXN associated with g-radiation had a close to additive effect, except for 1 mg/ml of DXR in combination with 40 cGy, in which a synergistic effect was observed. As DXR generates reactive oxygen species besides inhibiting topoisomerase II activity (Myers et al., 1977; Keizer et al., 1990), a stronger interaction between DXR and g-radiation when compared with MXN-g-radiation association was observed.

Although it did not show a synergistic effect according to statistical test, the combination of 0.04 mg/ml of MXN + 40 cGy of g-radiation increased the total number of chromosome aberrations and remained close to significance (^{Table II}). However, the low mitotic index found in combined treatments may have influenced our results, since a larger number of dead cells could mask the real data, since topoisomerase II inhibitors can trigger apoptosis (Lowe et al., 1993; Ferguson and Baguley, 1994).

Additive and possibly synergistic effects have also been reported in CHO cells treated with either 5-fluorouracil or hydroxyurea combined with radiation (Vokes et al., 1992), in HL-60 cells treated with taxol combined with radiation (Choy et al., 1993) and in cell lines derived from human carcinomas treated with taxol associated with radiation (Stromberg et al., 1995).

The mechanism of action of DNA topoisomerase II inhibitors has been investigated in a cell culture system (Anderson and Berger, 1994; Ferguson and Baguley, 1994). Other investigations have also been performed to evaluate the influence of drugs on radiation damage repair (O'Hara et al., 1986; Wallner and Li, 1987). Piro et al. (1976) reported that exponentially growing hamster cells in culture were less capable of repairing sublethal radiation damage if actinomycin-D was present in the medium at very low concentration between two X-ray exposures. Carboplatin was also shown to be an inhibitor of repair of potentially lethal radiation damage in V79 cells (O'Hara et al., 1986).

There is evidence that the enzyme topoisomerase II may be involved in the repair of radiation-induced DNA damage (Balosso et al., 1991). DXR and MXN are known to change DNA conformation by stabilization of the DNA topoisomerase II complex. Topoisomerase II inhibitors produce reversible protein-linked DNA breaks on cleavable complex (Bodley et al., 1987; Pommier and Bertrand, 1993). So, these inhibitors disrupt the breakage-reunion cycles of topoisomerases and produce single- and double-strand breaks (Snapka, 1987). According to Bonner and Lawrence (1990), it is possible that these changes alter the ability of repair enzymes to recognize and correct radiation-induced chromosomal damage. This could have happened in our test with 1 mg/ml of DXR combined with 40 cGy, in which a synergistic effect was observed.

Topoisomerase II inhibitors such as DXR and MXN have been found to efficiently induce chromosomal aberrations, mainly breaks and exchanges, in cultured Chinese hamster lung fibroblastic cells (Suzuki et al., 1995). In the present study, since CHO cultures were treated in the G2 phase of the cell cycle, chromatid breaks were the major type of damage observed, followed by gaps and exchanges. Chromatid breaks were present in large numbers, mainly in the combination in which a synergistic effect was noticed. The marked potentiation of the exchange- and chromatid break-type aberrations observed suggests that interaction between DXR- and radiation-induced lesions and the interference in the repair mechanism are the cause of the observed synergism.

Gaps are considered by some investigators to be of debatable genetic significance, because their presence does not always lead to chromosome aberrations in subsequent cell divisions (Preston et al., 1987; Brusick, 1987). Some authors include gaps in the general computation of aberrations (Goetz et al., 1975; Galloway et al., 1986; Dias et al., 1995), while others do not (Scheid and Traut, 1971; Brfgger, 1982). Based on these contradictory opinions, our data for DXR and MXN combined or not with g-radiation (Figures 1 and 2, respectively) were analyzed statistically including and excluding gaps. The results did not differ.

Figure 1
- Frequency of chromosomal aberrations in 300 cells with or without gaps produced by doxorubicin (DXR), g-radiation and combination of the two.

Figure 2
- Frequency of chromosomal aberrations in 300 cells with and without gaps produced by mitoxantrone (MXN), g-radiation and combinations of the two.

The additive effects observed and the synergistic effect that occurred with one specific combination may reflect a therapeutic benefit of combined treatment with DXR or MXN and g-radiation. However, the low mitotic index observed in combined treatments could be a limiting factor if its effects were greater on normal tissue than on tumor tissue. Further investigations would be necessary to evaluate the interactions between antineoplastic drugs and radiation, their cytotoxic effects and their efficacies in a clinical setting.

ACKNOWLEDGMENTS

The authors thank Farmitalia Carlo Erba do Brasil for donating doxorubicin, Ana Maria R. Romero for preparing mitoxantrone, Mr. Luiz A. Costa Jr. and Miss Sueli A. Neves for technical assistance, and Dr. Sergio Kronka for statistical help. Research supported by CAPES, FAPESP and CNPq. Publication supported by FAPESP.

RESUMO

A associação de radiação ionizante com drogas antineoplásicas tem importante aplicação na terapia do câncer. No presente trabalho, foram avaliados os efeitos de dois inibidores de topoisomerase II, doxorubicina (DXR) e mitoxantrona (MXN), sobre as aberrações cromossômicas induzidas pelas radiações-g em células do ovário de hamster chinês (CHO). Foram usadas as concentrações 0,5 e 1,0 mg/ml de DXR e 0,02 e 0,04 mg/ml de MXN, combinadas com duas doses de radiações gama (20 e 40 cGy). Um significativo efeito potenciador das aberrações cromossômicas foi observado em células CHO tratadas com 1,0 mg/ml de DXR e expostas a 40 cGy de radiação. Nos outros testes, a combinação da radiação-g com a DXR ou MXN apresentou um efeito próximo ao aditivo. A redução dos índices mitóticos refletiu a alta citotoxicidade das drogas quando combinadas às radiações-g.

(Received September 2, 1997)

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Publication Dates

• Publication in this collection
  23 Feb 1999
• Date of issue
  Sept 1998

History

• Received
  02 Sept 1997