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Effect of Concentration on the Cytotoxic Mechanism of Doxorubicin—Apoptosis and Oxidative DNA Damage
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
230, 254–257 (1997)
RC965898
Effect of Concentration on the Cytotoxic Mechanism of Doxorubicin—Apoptosis and Oxidative DNA Damage Ingo Mu¨ller,*,1 Andrew Jenner,† Gernot Bruchelt,* Dietrich Niethammer,* and Barry Halliwell† *Department of Haematology and Oncology, Children’s University Hospital, Ru¨melinstrasse 23, D-72070 Tu¨bingen, Germany; and †Neurodegenerative Disease Research Centre, King’s College, Pharmacology Group, Manresa Road, London SW3 6LX, United Kingdom
Received November 29, 1996
Anthracycline derivatives such as doxorubicin are part of many chemotherapeutic regimens and reach peak plasma concentrations of 5 mM. We investigated the cytotoxic mechanisms of various doxorubicin concentrations in MOLT-4 ALL-cells. Concentrations of up to 100 mM doxorubicin achieved similar cytotoxic effects in cultures of MOLT-4 cells, but acted via different mechanisms. Doxorubicin induced apoptosis (maximum effect at 1 mM), which was dependent on RNA synthesis and involved oxidative stress. Concentrations higher than 3 mM did not induce apoptosis, but significantly inhibited RNA synthesis. DNA strand breaks in MOLT-4 cells occurred in the presence of 1 to 5 mM doxorubicin to a similar extent, but showed a dose-dependence at higher concentrations. There was no GC/MS-detectable oxidation of DNA bases in apoptotic cells and only 1 out of 13 DNA base oxidation products, 8-hydroxyguanine, increased significantly in the presence of as much as 100 mM doxorubicin. These results suggest that at pharmacologically relevant concentrations apoptosis and not oxidative DNA damage is the main killing mechanism of doxorubicin against ALL-cells. q 1997 Academic Press
Acute lymphoblastic leukaemia (ALL) is the most frequent childhood leukaemia and several chemotherapeutic protocols achieve long-term remission in up to 85% of all cases (1). Often, these treatments include anthracycline derivatives such as doxorubicin. The cytostatic effect of these drugs can involve (i) inhibition of topoisomerase II and RNA polymerase II (2); (ii) intercalation into chromosomal DNA and formation of complexes with iron (3), provoking erroneous transcription and replication as well as generation of reactive
oxygen species (ROS). ROS can impair cell viability by damage to proteins, lipids, and DNA. (iii) ROS as well as anthracyclines themselves can induce apoptosis (5). Apoptosis is an active form of cell death characterized by distinct morphological changes and by internucleosomal DNA fragmentation. Important molecular features of the apoptotic process include engagement of CD95 (6), ceramide as a signaling molecule and interleukin-1b-converting enzyme-like proteases as executioners of the apoptotic program (7). Indeed, it is widely suggested, that ROS are not only able to induce apoptosis, but also to serve as an intracellular signal of the apoptotic cascade (8-10). By contrast, other groups reported, that apoptosis can occur even under low oxygen tension (11-13). Recently, it was shown that intracellular ceramide levels increased with daunorubicin doses and correlated with the numbers of apoptotic cells (14, 15). Ling et al. (5), however, reported that in their experimental setting 1 mM doxorubicin caused apoptosis, whereas 10 mM did not. These cytotoxic effects are relevant not only to anticancer activity, but also to side effects such as cardiomyopathy or nephrosis (16). One important intracellular target of anthracycline antibiotics is nuclear DNA. Therefore, we employed the single cell gel electrophoresis assay (SCG), in order to measure DNA strand breaks in individual cells (17). We also applied gas chromatography / mass spectrometry (GC/MS) techniques, determining oxidative damage to DNA bases in order to investigate the involvement of ROS in DNA damage of apoptotic cells (18). Our results show, that induction of apoptosis is the predominant mechanism of doxorubicin-mediated cytotoxicity at concentrations observed in blood plasma during chemotherapy. Doxorubicin-induced apoptosis was dependent on RNA synthesis. Oxidative damage was only observed at much higher doxorubicin concentrations. MATERIALS AND METHODS
1
Corresponding author. Fax: //49-7071-294448. E-mail: ingo. [email protected].
Cells and cell culture. MOLT-4 cells were obtained from the American Type Tissue Culture Collection (No. 1582) and cultured at
254
0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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Vol. 230, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
377C under 5% CO2 in RPMI 1640 supplemented with 10% (v/v) fetal calf serum, 0.5 mM L-glutamine, 100 I.E./ml penicillin, and 100 I.E./ ml streptomycin. All experiments were carried out with cells from exponentially growing cultures never exceeding a density of 5r105 cells/ml. Actinomycin D (Calbiochem) and butylated hydroxyanisole (Sigma) were added to MOLT-4 cell cultures 90 min prior to application of 1 mM doxorubicin for 12 hours. Single cell gel electrophoresis. Single and double strand breaks in nuclear DNA were detected in the alkaline version of the SCG assay, which was modified according to Singh et al. (19). Fully frosted microscope slides (Richardson, UK) were covered with 1%(w/ v) agarose. 104 cells were suspended in 0.5% LGT-agarose (Sigma), transferred onto the first agarose layer and covered with a cover slip. After solidification, the cover slip was removed and another layer of 0.5% LGT-agarose loaded onto the slide. For cell lysis, the microscope slides were submerged in an ice-cold solution of 2.5 M NaCl, 100 mM EDTA, 1%(w/v) N-lauroylsarcosine, 10 mM TRIS (pH 10) and freshly added 1%(v/v) Triton X-100 and 10%(v/v) DMSO. After incubation for one hour, the digestion buffer was replaced by an alkaline solution (300 mM NaOH and 1 mM EDTA) to allow unwinding of the DNA strands for 30 min on ice. The microscope slides are transferred to the pre-cooled plate of the electrophoresis system Multiphor II (Pharmacia) and subjected to electrophoresis for 15 min at 47C with 10 mA per microscope slide, typically requiring an electric force field of 3 V/cm. Microgels are neutralized with 0.4 M TRIS before staining with an aqueous solution of 50 mg/ml propidium iodide. Nuclei were evaluated by fluorescence microscopy with an excitation wavelength of 515-550 nm and divided into four classes (0 through 3), according to length and intensity of nucleus and tail (20). The damage index was calculated by multiplication of the percentage of nuclei in each damage class with the number (0 through 3) of this class. Hence, if all nuclei are undamaged (100% class 0) the damage index is 0 and if 100% of all scored nuclei belong to class 3, the damage index is 300. Cell death assays. During apoptotic cell death internucleosomal DNA fragmentation occurs and nucleosomes are liberated into the cytoplasm, which can be measured by the cell death detection ELISA (Boehringer Mannheim, Germany). The ELISA was performed according to the supplier’s instructions. In addition, characteristic morphological changes associated with apoptosis were investigated by fluorescence microscopy of propidium iodide stained cells. 105 cells were incubated in ice-cold 70%(v/v) ethanol for one hour and fixed on a microscopy slide by cytocentrifugation. Subsequently, cells were covered with a solution of 50 mg/ml propidium iodide and 0.1%(w/v) ribonuclease A in PBS, pH 7.4. After washing with PBS, preparations were analyzed with an excitation wavelength of 515-560 nm on a fluorescence microscope.
FIG. 1. Growth inhibition of MOLT-4 ALL-cells by various concentrations of doxorubicin.
FIG. 2. DNA strand breaks induced by doxorubicin assessed by single cell gel electrophoresis. The damage index is a measure of the number of single and double strand breaks (see Materials and Methods).
RNA synthesis. MOLT-4 cells were incubated with various concentrations of doxorubicin for 12 hours. Subsequently, 5 mCi [5,63 H]-uridine were added to 106 MOLT-4 cells. Incorporation at 377C was stopped after 15 min with 10 ml ice-cold PBS. Cells were harvested and lysed in 5 ml 5%(w/v) TCA on ice for 30 min. The lysate was centrifuged at 2000 rpm and the pellet redissolved in 500 ml 0.2 M NaOH. 400 ml of this solution were assayed for incorporated [5,63 H]-uridine in a liquid scintillation counter. DNA isolation and analysis of oxidative damage by gas chromatography/mass spectrometry. Sample preparation was carried out as described previously (18) using DNA from 2.5r107 cells.
RESULTS AND DISCUSSION Initially, we analyzed the dose-dependence of doxorubicin-induced cytotoxicity in vitro. Unexpectedly, concentrations between 1 and 100 mM achieved similar cytostatic effects in cultures of the ALL-cell line MOLT4 (Fig. 1). This was accompanied by DNA strand breakage (Fig. 2). After a plateau in the presence of 1 to 5 mM doxorubicin, a discrete decrease in the range from
FIG. 3. Apoptosis in cultures of MOLT-4 cells incubated with various concentrations of doxorubicin for 12 hours (measured by the cell death detection ELISA (Boehringer Mannheim, Germany), where the absorbance at 490 nm is proportional to the number of apoptotic cells). The results were verified by morphological analysis of cytospin preparations.
255
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12-24-96 00:23:12
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AP: BBRC
Vol. 230, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 4. Dependence of doxorubicin-induced apoptosis on RNA synthesis. (a) RNA synthesis in MOLT-4 cells incubated with doxorubicin for 12 hours, as determined by [3H]-uridine incorporation into RNA. (b) Abrogation of doxorubicin-induced apoptosis by pre-incubation with actinomycin D for 90 min.
5 to 50 mM was followed by a dose-dependent increase of DNA strand breaks at even higher doses of doxorubicin. This suggested different mechanisms of DNA damage depending on the doxorubicin concentration. We used two independent experimental approaches to detect apoptosis in MOLT-4 cells incubated with doxorubicin. Figure 3 shows that after 12 hours of incubation internucleosomal DNA fragmentation was detectable only in the presence of up to 3 mM doxorubicin. Remarkably, higher concentrations were not capable of committing these ALL-cells to apoptotic death, but rather to cell death lacking internucleosomal DNA fragmentation, formation of apoptotic bodies etc. All these results were confirmed by morphological analysis (data not shown). Doxorubicin is known to inhibit chicken myeloblastosis RNA polymerase II (2) and cycloheximide, an inhibitor of protein synthesis, blocks anthracycline-induced apoptosis in vivo (21). Based on these findings,
FIG. 5. The antiooxidant butylated hydroxyanisole inhibits doxorubicin-induced apoptosis. MOLT-4 cells were pre-incubated with the antioxidant for 90 min, before 1 mM doxorubicin was added.
we speculated that doxorubicin concentrations ú 1 mM increasingly inhibit de novo gene expression of proteins, e. g. FasL, necessary for induction of the apoptotic program (6), whereas 1 mM doxorubicin still allows for transcription of these genes and induces apoptosis. Figure 4a) shows that the presence of 10 mM or 100 mM doxorubicin in cultures of MOLT-4 cells significantly inhibited RNA synthesis after 12 hours of incubation. In contrast, RNA synthesis of MOLT-4 cells incubated with 1 mM doxorubicin was only slightly reduced during this period of time. Thus, although doxorubicin concentrations ú 1 mM might still be able to deliver an apoptotic signal, a second activity in this concentration range, inhibition of RNA synthesis, interferes with the apoptotic process. This view was further supported by the suppression of doxorubicin-induced apoptosis in the presence of actinomycin D, a
FIG. 6. Oxidative damage of DNA bases determined by GC/MS. The content of 8-hydroxyguanine in nuclear DNA isolated from MOLT-4 cells after incubation with doxorubicin for 12 hours was not elevated in apoptotic cells (1 mM doxorubicin), only in cells treated with much higher doses of the anthracycline.
256
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12-24-96 00:23:12
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AP: BBRC
Vol. 230, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
potent inhibitor of transcription (Fig. 4b), and puromycin, an inhibitor of translation (data not shown). Since anthracyclines can induce oxidative stress, we examined the effect of the antioxidant butylated hydroxyanisole on doxorubicin-induced apoptosis. Fig. 5 shows that it efficiently inhibited doxorubicin-induced apoptosis. As oxidative stress can cause DNA damage and we used GC/MS to detect 14 different oxidation products of DNA bases (18). Only one of these, 8-hydroxyguanine, displayed any changes. MOLT-4 cells showed no increase in their 8-hydroxyguanine content when incubated with 1 mM or 10 mM doxorubicin for 12 hours (Fig. 6), i. e. oxidative damage was here not involved in apoptosis. By contrast, the presence of 100 mM doxorubicin caused an almost threefold increase of 8-hydroxyguanine in the DNA. Taken together, our experiments confirmed that doxorubicin-induced apoptosis in MOLT-4 cells with a maximum at a concentration of 1 mM, but not at concentrations higher than 3 mM. Recently, Bose and co-workers (15) reported that daunorubicin induced apoptosis in P388 and U937 cells is due to elevation of ceramide synthesis. In this system, ceramide concentrations increased with increasing doses of the anthracycline derivative. However, in our experiments, high concentrations of doxorubicin (ú1 mM) did not result in more abundant apoptotic cell death. We suggest that different cytotoxic activities of doxorubicin interfere with each other. At doses up to 3 mM doxorubicin predominantly acts via induction of apoptosis. At higher concentrations, the apoptotic stimulus might still be initiated (perhaps involving ceramide), but the apoptotic program cannot be completed, because the synthesis of proteins (e. g. FasL etc.), is inhibited at the level of transcription (Fig. 4). Our data suggest that at concentrations of 100 mM and more, doxorubicin exerts its cytostatic mechanism not only by inhibition of RNA polymerases, but also by oxidative damage (Fig. 2 and 5). Doxorubicin concentrations higher than 5 mM have never been reported during therapy in vivo, hence apoptosis and not oxidative DNA damage is likely to be the predominant cytotoxic mechanism of doxorubicin in leukaemia treatment (22). Further experiments aim at the elucidation of the role of ceramide synthesis and oxidative stress in the doxorubicin-derived apoptotic stimulus.
ACKNOWLEDGMENT This work was supported in part by the Evangelisches Studienwerk Villigst e.V.
REFERENCES 1. Pui, C. H., and Crist, W. M. (1995) Curr. Opin. Oncol. 7, 36–44. 2. Chuang, R. Y., and Chuang, L .F. (1979) Biochemistry 18, 2069– 2073. 3. Neidle, S., and Sanderson, M. R. (1983) in Molecular Aspects of Anti-Cancer Drug Action (Neidle, S., and Waring, M. J., Eds.), pp. 35–53, Verlag Chemie, Weinheim, Germany. 4. Cheng, K. C., Cahill, D. S., Kasai, H., Nishimura, S., and Loeb, L. A. (1992) J. Biol. Chem. 267, 166–172. 5. Ling, Y.-H., Priebe, W., and Perez-Soler, R. (1993) Cancer Res. 53, 1845–1852. 6. Friesen, C., Herr, I., Krammer, P. H., and Debatin, K.-M. (1996) Nature Med. 2, 574–577. 7. Hannun, Y. A., and Obeid, L. M. (1995) Trends Biol. Sci. 20, 73– 77. 8. Buttke, T. M., and Sanstrom, P. A. (1994) Immunol. Tod. 15, 7– 10. 9. Hockenbery, D., Oltvai, Z. N., Yin, X., Milliman, C. L., and Korsmeyer, S. J. (1993) Cell 75, 241–251. 10. Slater, A. F., Nobel, C. S., and Orrenius, S. (1995) Biochim. Biophys. Acta 1271, 59–62. 11. Muschel, R. J., Bernhard, E. J., Garza, L., McKenna, W. G., and Koch, C. J. (1995) Cancer Res. 55, 995–998. 12. Jacobson, M. D., and Raff, M. C. (1995) Nature 374, 814–816. 13. Shimizu, S., Eguchi, Y., Kosaka, H., Kamiike, W., Matsuda, H., and Tsujimoto, Y. (1995) Nature 374, 811–813. 14. Jaffre`zou, J.-P., Levade, T., Bettaieb, A., Andrieu, N., Bezombes, C., Maestre, N., Vermeersch, S., Rousse, A., and Laurent, G. (1996) Embo J. 15, 2417–2424. 15. Bose, R., Verheij, M., Haimovitz-Friedmann, A., Scotto, K., Fuks, Z., and Kolesnick, R. (1995) Cell 82, 405–414. 16. Halliwell, B., and Bomford, A. (1989) N. Engl. J. Med. 320, 399– 400. 17. Fairbairn, D. W., Olive, P. L., and O’Neill, K. L. (1995) Mutation Res. 339, 37–59. 18. Spencer, J. P. E., Jenner, A., Chimel, K., Aruoma, O. I., Cross, C. E., Wu, R., and Halliwell, B. (1995) FEBS Lett. 374, 233–236. 19. Singh, N. P., McCoy M. T., Tice, R. R., and Schneider, E. L. (1988) Exp. Cell. Res. 175, 184–191. 20. Gedik, C. M., Ewen, S. W. B., and Collins, A. R. (1992) Int. J. Radiat. Biol. 62, 313–320. 21. Thakkar, N. S., and Potten, C. S. (1992) Biochem. Pharmacol. 43, 1683–1691. 22. Gorczyca, W., Bigman, K., Mittelman, A., Ahmed, T., Gong, J., Melamed, M. R., and Darzynkiewicz, Z. (1993) Leukemia 7, 659– 670.
257
AID
BBRC 5898
/
6916$$$101
12-24-96 00:23:12
bbrcgs
AP: BBRC
230, 254–257 (1997)
RC965898
Effect of Concentration on the Cytotoxic Mechanism of Doxorubicin—Apoptosis and Oxidative DNA Damage Ingo Mu¨ller,*,1 Andrew Jenner,† Gernot Bruchelt,* Dietrich Niethammer,* and Barry Halliwell† *Department of Haematology and Oncology, Children’s University Hospital, Ru¨melinstrasse 23, D-72070 Tu¨bingen, Germany; and †Neurodegenerative Disease Research Centre, King’s College, Pharmacology Group, Manresa Road, London SW3 6LX, United Kingdom
Received November 29, 1996
Anthracycline derivatives such as doxorubicin are part of many chemotherapeutic regimens and reach peak plasma concentrations of 5 mM. We investigated the cytotoxic mechanisms of various doxorubicin concentrations in MOLT-4 ALL-cells. Concentrations of up to 100 mM doxorubicin achieved similar cytotoxic effects in cultures of MOLT-4 cells, but acted via different mechanisms. Doxorubicin induced apoptosis (maximum effect at 1 mM), which was dependent on RNA synthesis and involved oxidative stress. Concentrations higher than 3 mM did not induce apoptosis, but significantly inhibited RNA synthesis. DNA strand breaks in MOLT-4 cells occurred in the presence of 1 to 5 mM doxorubicin to a similar extent, but showed a dose-dependence at higher concentrations. There was no GC/MS-detectable oxidation of DNA bases in apoptotic cells and only 1 out of 13 DNA base oxidation products, 8-hydroxyguanine, increased significantly in the presence of as much as 100 mM doxorubicin. These results suggest that at pharmacologically relevant concentrations apoptosis and not oxidative DNA damage is the main killing mechanism of doxorubicin against ALL-cells. q 1997 Academic Press
Acute lymphoblastic leukaemia (ALL) is the most frequent childhood leukaemia and several chemotherapeutic protocols achieve long-term remission in up to 85% of all cases (1). Often, these treatments include anthracycline derivatives such as doxorubicin. The cytostatic effect of these drugs can involve (i) inhibition of topoisomerase II and RNA polymerase II (2); (ii) intercalation into chromosomal DNA and formation of complexes with iron (3), provoking erroneous transcription and replication as well as generation of reactive
oxygen species (ROS). ROS can impair cell viability by damage to proteins, lipids, and DNA. (iii) ROS as well as anthracyclines themselves can induce apoptosis (5). Apoptosis is an active form of cell death characterized by distinct morphological changes and by internucleosomal DNA fragmentation. Important molecular features of the apoptotic process include engagement of CD95 (6), ceramide as a signaling molecule and interleukin-1b-converting enzyme-like proteases as executioners of the apoptotic program (7). Indeed, it is widely suggested, that ROS are not only able to induce apoptosis, but also to serve as an intracellular signal of the apoptotic cascade (8-10). By contrast, other groups reported, that apoptosis can occur even under low oxygen tension (11-13). Recently, it was shown that intracellular ceramide levels increased with daunorubicin doses and correlated with the numbers of apoptotic cells (14, 15). Ling et al. (5), however, reported that in their experimental setting 1 mM doxorubicin caused apoptosis, whereas 10 mM did not. These cytotoxic effects are relevant not only to anticancer activity, but also to side effects such as cardiomyopathy or nephrosis (16). One important intracellular target of anthracycline antibiotics is nuclear DNA. Therefore, we employed the single cell gel electrophoresis assay (SCG), in order to measure DNA strand breaks in individual cells (17). We also applied gas chromatography / mass spectrometry (GC/MS) techniques, determining oxidative damage to DNA bases in order to investigate the involvement of ROS in DNA damage of apoptotic cells (18). Our results show, that induction of apoptosis is the predominant mechanism of doxorubicin-mediated cytotoxicity at concentrations observed in blood plasma during chemotherapy. Doxorubicin-induced apoptosis was dependent on RNA synthesis. Oxidative damage was only observed at much higher doxorubicin concentrations. MATERIALS AND METHODS
1
Corresponding author. Fax: //49-7071-294448. E-mail: ingo. [email protected].
Cells and cell culture. MOLT-4 cells were obtained from the American Type Tissue Culture Collection (No. 1582) and cultured at
254
0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 5898
/
6916$$$101
12-24-96 00:23:12
bbrcgs
AP: BBRC
Vol. 230, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
377C under 5% CO2 in RPMI 1640 supplemented with 10% (v/v) fetal calf serum, 0.5 mM L-glutamine, 100 I.E./ml penicillin, and 100 I.E./ ml streptomycin. All experiments were carried out with cells from exponentially growing cultures never exceeding a density of 5r105 cells/ml. Actinomycin D (Calbiochem) and butylated hydroxyanisole (Sigma) were added to MOLT-4 cell cultures 90 min prior to application of 1 mM doxorubicin for 12 hours. Single cell gel electrophoresis. Single and double strand breaks in nuclear DNA were detected in the alkaline version of the SCG assay, which was modified according to Singh et al. (19). Fully frosted microscope slides (Richardson, UK) were covered with 1%(w/ v) agarose. 104 cells were suspended in 0.5% LGT-agarose (Sigma), transferred onto the first agarose layer and covered with a cover slip. After solidification, the cover slip was removed and another layer of 0.5% LGT-agarose loaded onto the slide. For cell lysis, the microscope slides were submerged in an ice-cold solution of 2.5 M NaCl, 100 mM EDTA, 1%(w/v) N-lauroylsarcosine, 10 mM TRIS (pH 10) and freshly added 1%(v/v) Triton X-100 and 10%(v/v) DMSO. After incubation for one hour, the digestion buffer was replaced by an alkaline solution (300 mM NaOH and 1 mM EDTA) to allow unwinding of the DNA strands for 30 min on ice. The microscope slides are transferred to the pre-cooled plate of the electrophoresis system Multiphor II (Pharmacia) and subjected to electrophoresis for 15 min at 47C with 10 mA per microscope slide, typically requiring an electric force field of 3 V/cm. Microgels are neutralized with 0.4 M TRIS before staining with an aqueous solution of 50 mg/ml propidium iodide. Nuclei were evaluated by fluorescence microscopy with an excitation wavelength of 515-550 nm and divided into four classes (0 through 3), according to length and intensity of nucleus and tail (20). The damage index was calculated by multiplication of the percentage of nuclei in each damage class with the number (0 through 3) of this class. Hence, if all nuclei are undamaged (100% class 0) the damage index is 0 and if 100% of all scored nuclei belong to class 3, the damage index is 300. Cell death assays. During apoptotic cell death internucleosomal DNA fragmentation occurs and nucleosomes are liberated into the cytoplasm, which can be measured by the cell death detection ELISA (Boehringer Mannheim, Germany). The ELISA was performed according to the supplier’s instructions. In addition, characteristic morphological changes associated with apoptosis were investigated by fluorescence microscopy of propidium iodide stained cells. 105 cells were incubated in ice-cold 70%(v/v) ethanol for one hour and fixed on a microscopy slide by cytocentrifugation. Subsequently, cells were covered with a solution of 50 mg/ml propidium iodide and 0.1%(w/v) ribonuclease A in PBS, pH 7.4. After washing with PBS, preparations were analyzed with an excitation wavelength of 515-560 nm on a fluorescence microscope.
FIG. 1. Growth inhibition of MOLT-4 ALL-cells by various concentrations of doxorubicin.
FIG. 2. DNA strand breaks induced by doxorubicin assessed by single cell gel electrophoresis. The damage index is a measure of the number of single and double strand breaks (see Materials and Methods).
RNA synthesis. MOLT-4 cells were incubated with various concentrations of doxorubicin for 12 hours. Subsequently, 5 mCi [5,63 H]-uridine were added to 106 MOLT-4 cells. Incorporation at 377C was stopped after 15 min with 10 ml ice-cold PBS. Cells were harvested and lysed in 5 ml 5%(w/v) TCA on ice for 30 min. The lysate was centrifuged at 2000 rpm and the pellet redissolved in 500 ml 0.2 M NaOH. 400 ml of this solution were assayed for incorporated [5,63 H]-uridine in a liquid scintillation counter. DNA isolation and analysis of oxidative damage by gas chromatography/mass spectrometry. Sample preparation was carried out as described previously (18) using DNA from 2.5r107 cells.
RESULTS AND DISCUSSION Initially, we analyzed the dose-dependence of doxorubicin-induced cytotoxicity in vitro. Unexpectedly, concentrations between 1 and 100 mM achieved similar cytostatic effects in cultures of the ALL-cell line MOLT4 (Fig. 1). This was accompanied by DNA strand breakage (Fig. 2). After a plateau in the presence of 1 to 5 mM doxorubicin, a discrete decrease in the range from
FIG. 3. Apoptosis in cultures of MOLT-4 cells incubated with various concentrations of doxorubicin for 12 hours (measured by the cell death detection ELISA (Boehringer Mannheim, Germany), where the absorbance at 490 nm is proportional to the number of apoptotic cells). The results were verified by morphological analysis of cytospin preparations.
255
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6916$$$101
12-24-96 00:23:12
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AP: BBRC
Vol. 230, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 4. Dependence of doxorubicin-induced apoptosis on RNA synthesis. (a) RNA synthesis in MOLT-4 cells incubated with doxorubicin for 12 hours, as determined by [3H]-uridine incorporation into RNA. (b) Abrogation of doxorubicin-induced apoptosis by pre-incubation with actinomycin D for 90 min.
5 to 50 mM was followed by a dose-dependent increase of DNA strand breaks at even higher doses of doxorubicin. This suggested different mechanisms of DNA damage depending on the doxorubicin concentration. We used two independent experimental approaches to detect apoptosis in MOLT-4 cells incubated with doxorubicin. Figure 3 shows that after 12 hours of incubation internucleosomal DNA fragmentation was detectable only in the presence of up to 3 mM doxorubicin. Remarkably, higher concentrations were not capable of committing these ALL-cells to apoptotic death, but rather to cell death lacking internucleosomal DNA fragmentation, formation of apoptotic bodies etc. All these results were confirmed by morphological analysis (data not shown). Doxorubicin is known to inhibit chicken myeloblastosis RNA polymerase II (2) and cycloheximide, an inhibitor of protein synthesis, blocks anthracycline-induced apoptosis in vivo (21). Based on these findings,
FIG. 5. The antiooxidant butylated hydroxyanisole inhibits doxorubicin-induced apoptosis. MOLT-4 cells were pre-incubated with the antioxidant for 90 min, before 1 mM doxorubicin was added.
we speculated that doxorubicin concentrations ú 1 mM increasingly inhibit de novo gene expression of proteins, e. g. FasL, necessary for induction of the apoptotic program (6), whereas 1 mM doxorubicin still allows for transcription of these genes and induces apoptosis. Figure 4a) shows that the presence of 10 mM or 100 mM doxorubicin in cultures of MOLT-4 cells significantly inhibited RNA synthesis after 12 hours of incubation. In contrast, RNA synthesis of MOLT-4 cells incubated with 1 mM doxorubicin was only slightly reduced during this period of time. Thus, although doxorubicin concentrations ú 1 mM might still be able to deliver an apoptotic signal, a second activity in this concentration range, inhibition of RNA synthesis, interferes with the apoptotic process. This view was further supported by the suppression of doxorubicin-induced apoptosis in the presence of actinomycin D, a
FIG. 6. Oxidative damage of DNA bases determined by GC/MS. The content of 8-hydroxyguanine in nuclear DNA isolated from MOLT-4 cells after incubation with doxorubicin for 12 hours was not elevated in apoptotic cells (1 mM doxorubicin), only in cells treated with much higher doses of the anthracycline.
256
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12-24-96 00:23:12
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AP: BBRC
Vol. 230, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
potent inhibitor of transcription (Fig. 4b), and puromycin, an inhibitor of translation (data not shown). Since anthracyclines can induce oxidative stress, we examined the effect of the antioxidant butylated hydroxyanisole on doxorubicin-induced apoptosis. Fig. 5 shows that it efficiently inhibited doxorubicin-induced apoptosis. As oxidative stress can cause DNA damage and we used GC/MS to detect 14 different oxidation products of DNA bases (18). Only one of these, 8-hydroxyguanine, displayed any changes. MOLT-4 cells showed no increase in their 8-hydroxyguanine content when incubated with 1 mM or 10 mM doxorubicin for 12 hours (Fig. 6), i. e. oxidative damage was here not involved in apoptosis. By contrast, the presence of 100 mM doxorubicin caused an almost threefold increase of 8-hydroxyguanine in the DNA. Taken together, our experiments confirmed that doxorubicin-induced apoptosis in MOLT-4 cells with a maximum at a concentration of 1 mM, but not at concentrations higher than 3 mM. Recently, Bose and co-workers (15) reported that daunorubicin induced apoptosis in P388 and U937 cells is due to elevation of ceramide synthesis. In this system, ceramide concentrations increased with increasing doses of the anthracycline derivative. However, in our experiments, high concentrations of doxorubicin (ú1 mM) did not result in more abundant apoptotic cell death. We suggest that different cytotoxic activities of doxorubicin interfere with each other. At doses up to 3 mM doxorubicin predominantly acts via induction of apoptosis. At higher concentrations, the apoptotic stimulus might still be initiated (perhaps involving ceramide), but the apoptotic program cannot be completed, because the synthesis of proteins (e. g. FasL etc.), is inhibited at the level of transcription (Fig. 4). Our data suggest that at concentrations of 100 mM and more, doxorubicin exerts its cytostatic mechanism not only by inhibition of RNA polymerases, but also by oxidative damage (Fig. 2 and 5). Doxorubicin concentrations higher than 5 mM have never been reported during therapy in vivo, hence apoptosis and not oxidative DNA damage is likely to be the predominant cytotoxic mechanism of doxorubicin in leukaemia treatment (22). Further experiments aim at the elucidation of the role of ceramide synthesis and oxidative stress in the doxorubicin-derived apoptotic stimulus.
ACKNOWLEDGMENT This work was supported in part by the Evangelisches Studienwerk Villigst e.V.
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