Inr. 1. Radiation Oncology Biol. Phyr., Vol. 22. pp. 519-523 Pnnted in the U S.A. All rights reserved.
0360-3016/92 $5.00 + .oO Q 1992 Pergamon &ss plc
??Session B: Biochemical Modification of Therapeutic Response
INTERACTION OF BUTHIONINE SULFOXIMINE AND THE STABILIZATION OF DNA-TOPOISOMERASE II COMPLEXES BY DOXORUBICIN JAMES A. BONNER,
M.D. ,l TERESA J. H. CHRISTIANSON, B. S. ’ PH.D.*
AND THEODORE S. LAWRENCE, M.D.,
‘Mayo Clinic, Radiation Oncology, Rochester, MN; and ‘University of Michigan, Radiation Oncology, Ann Arbor, MI Although it has been shown previously that the depletion of cellular thiols increases doxorubicin cytotoxicity, the mechanism of sensitization is not clear. To study this question, the effect of D,L-butbionine-S,R-sulfoximine (BSO) on doxorubicin cytotoxicity and the stabilization of DNA-topoisomerase II complexes (cleavable complexes) was investigated in V79 ceils. Incubations with BSO (10 mM) were for 5 hr beginning 4 hr prior to doxorubicin exposure since a 4 hr incubation with 10 mM BSO is known to decrease glutathione levels below 5% of control V79 cells. These BSO pre-treatments increased doxorubicin cytotoxicity. At doxorubicin concentrations of 5 pg/ml, BSO resulted in an g-10 fold decrease in surviving cells, compared to cells exposed to doxorubicin alone. It was determined that BSO pre-treatments did not affect the accumulation of doxorubicin into the cell, the rate of cleavable complex stabilization by doxorubicin, or the rate of dissociation of stabilized cleavable complexes. These data suggest that BSO-induced doxorubicin sensitization occurs at a step following the stabilization of cleavable complexes or by an independent mechanism. Doxorubicin, Buthionine sulfoximine, Topoisomerase. INTRODUCTION
nine sulfoximine (BSO), a specific inhibitor of y-glutamylcysteine synthetase. Studies using BSO to lower GSH levels in doxorubicin-resistant MCF-7 breast tumor cells and control breast tumor cells have suggested an association between doxorubicin resistance and the cells capacity to detoxify free radicals through GSH (3). The interaction of doxorubicin-induced free radicals and the stabilization of cleavable complexes by doxorubicin is not known. It has been previously determined that BSO pre-treatments (in concentrations and exposure times known to deplete GSH) result in sensitization of doxorubitin cytotoxicity in V79 cells (7). It seemed possible that greater free radical activity in the presence of BSO may increase doxorubicin sensitivity by affecting enzymatic or conformational requirements for doxorubicin to stabilize cleavable complexes. Therefore, an investigation was performed to determine if BSO pre-treatments potentiated doxorubicin cytotoxicity through the modulation of stabilized DNA-topoisomerase II complexes or alternatively through an independent process.
The chemotherapeutic agent doxorubicin is widely used for adult and childhood malignancies, but its mechanism of action remains controversial. Contributing to this controversy is the fact that doxorubicin is known to have various biochemical interactions within the cell, but the sequence of interactions required for cell lethality is unknown. Much evidence now exists that the stabilization of complexes between the enzyme topoisomerase II and DNA (cleavable complexes) by doxorubicin is necessary, but not sufficient for cell lethality (13). Although it has been hypothesized that certain proteins may be involved in cell lethality after the stabilization of cleavable complexes by doxorubicin (2, 8), the events following cleavable complex stabilization are not determined. In addition to doxorubicin’s interactions with the enzyme topoisomerase II, it is also known to undergo reduction to a semiquinone free radical, which subsequently results in the formation of hydroxyl free radicals (9). It has been proposed that these hydroxyl radicals lead to cell death by damage to critical cellular lipids or DNA (10). Cellular responses to free radicals are modified by glutathione (GSH), a naturally occuring sulfhydry-containing tripeptide that is known to detoxify free radicals. It is possible to examine the role of GSH with respect to doxorubicin cytotoxicity by lowering GSH levels with buthio-
Cell culture Hamster lung fibroblasts (V79) were kindly provided by James Mitchell, Ph.D., and were cultured in DMEM me-
Reprint requests to: James A. Bonner, M.D., Mayo Clinic CH-R, Department of Radiation Oncology, Rochester, MN
55905. Accepted for publication 3 July 1991. 519
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dium supplemented with penicillin, streptomycin, and 15% fetal bovine serum that was heat inactivated at 56°C for 1 hr. Cell lines were maintained at 37°C in a humidified atmosphere of 5% CO, and 95% air.
Drug treatment BSO was made fresh for each experiment. Doxorubicin hydrochloride* was made as a 500 pg/ml stock in phosphate-buffered saline (PBS), aliquotted, frozen at - 7O”C, and diluted on the day of the experiment. Cell survival Cells were treated with the appropriate drug combinations, removed from the dish with PBS containing 0.03% trypsin and 0.27 mM EDTA, and diluted into culture dishes in numbers to yield 40-200 colonies. Dilutions were performed in triplicate. After 6-7 days, cultures were fixed with methanol-acetic acid, stained with crystal violet, and scored for colonies containing more than 50 cells. For this assay, all experiments were repeated at least three times, but representative experiments are shown. Individual experiments were performed in triplicate and standard errors were typically less than 15% of the mean and are contained within the size of the symbol unless otherwise stated.
Determination of doxorubicin influx and efJlux Cells were exposed to drug treatment, washed with PBS, removed from the dish with trypsin containing 10 mM azide, washed with ice cold PBS containing 10 mM azide, and extracted with 2 ml of 0.3 NHC1:48% ethanol solution. Fluorescence spectrophotometry was used to determine doxorubicin content (excitation 470 nm; fluorescence 585 nm), as previously described (1) with minor modifications (6). Data were expressed as ng of doxorubitin per million cells. For this assay all experiments were repeated at least three times, but representative experiments are shown. Individual experiments were performed in triplicate and standard errors for these triplicate samples are shown.
Determination of stabilized DNA-topoisomerase II complexes Complexes were detected as single strand breaks using alkaline elution under conditions of proteolysis with proteinase K, as described previously (5) with minor modifications (6). Elutions were carried out at a rate of 0.04 ml/min using an internal standard irradiated with 1.0 Gy. Doxorubicin-induced DNA strand breakage was expressed as equivalent to a dose of radiation that yields the same number of strand breaks (rad equivalents). It should be noted that the alkaline elution procedure is a nonspecific procedure for measuring DNA-protein complexes. However, when proper controls are used (6), it is a well estab*Obtained as freeze-dried tories, Columbus, OH.
powder with lactose, Adria Labora-
Fig. 1. Buthionine sulfoximine sensitizes V79 cells to doxorubitin cytotoxicity. V79 cells were exposed to increasing concentrations of doxorubicin for 1 hr in the absence (closed squares) or the presence (open squares) of buthionine sulfoximine (10 mM). Incubations with buthionine sulfoximine (10 n&l) were for 5 hr beginning 4 hr prior to doxorubicin exposure. Cells were then assessed for cell survival as described in the Methods and Materials.
lished technique for the comparison of DNA-topoisomerase II complexes stabilized by doxorubicin (13). RESULTS To investigate the effect of glutathione depletion on doxorubicin sensitivity, V79 cells were exposed to increasing concentrations of doxorubicin for 1 hr in the absence or presence of BSO. Incubations with BSO (10 mM) were for 5 hr beginning 4 hr prior to doxorubicin exposure because it has been previously determined that a 4 hr incubation with 10 mM BSO decreases glutathione levels below 5% of control values and results in increased doxorubicin sensitivity in V79 cells (7). It was confirmed that V79 cells exposed to BSO pre-treatments were more sensitive to doxorubicin than cells exposed to doxorubicin alone (Fig. 1). At doxorubicin concentrations of 5 pg/ml, BSO resulted in an 8-10 fold decrease in surviving cells, compared to the surviving fraction resulting after doxorubicin exposures alone. Two possible mechanisms for sensitization were investigated. First, the effect of BSO on doxorubicin influx and efflux was assessed. It seemed possible that increased free radical production in the presence of BSO may alter carrier proteins, which are known to be responsible for doxorubicin accumulation (11). The influx and efflux of doxorubicin were examined under conditions identical to cell survival. When cells were incubated with 10 mM BSO for 4 hr prior to doxorubicin exposure for various time intervals, no difference in intracellular doxorubicin accumu-
and BSO 0 J. A. BONNERet al
Fig. 2. Buthionine sulfoximine does not affect doxorubicin influx into the cell. V79 cells were exposed to buthionine sulfoximine (10 mM) for 4 hr prior to a co-incubation with doxorubicin (4 pg/ml) for various time periods. Control V79 cells were exposed to doxorubicin (4 kg/ml) alone for various time periods. After completion of the doxorubicin exposure, cells were assessed for doxorubicin content as described in Methods and Materials.
efflux of doxorubicin was rubicin alone (Fig. 2). Similarly, not affected when cells were pre-treated with BSO beginning 4 hr prior to a 1 hr co-incubation with doxorubicin, followed by various efflux times in medium with BSO (Fig. 3). Since doxorubicin accumulation was unaffected by BSO, it was then of interest to determine whether BSO
Fig. 3. Buthionine sulfoximine does not affect doxorubicin efflux from the cell. V79 cells were exposed to 4 hr of buthionine sulfoximine (10 n&l), a 1 hr coincubation with buthionine sulfoximine and doxorubicin (5 kg/ml) followed by various postincubation times with buthionine sulfoximide alone. Control cells were exposed to doxorubicin alone for 1 hr followed by various post-incubation times in medium. Following the appropriate efflux time, cells were assessed for doxorubicin content as described in Methods and Materials.
Fig. 4. Buthionine sulfoximine does not affect the rate of stabilization of cleavable complexes by doxorubicin. V79 cells were exposed to buthionine sulfoximine (10 mM) for 4 hr prior to a co-incubation with buthionine sulfoximine and doxorubicin (5 pg/ml) for various time periods. The stabilization of cleavable complexes was assessed as described in Methods and Materials. Means and standard errors of three experiments are shown.
treatments altered the stabilization of cleavable complexes by doxorubicin. Consideration was given to the fact that increased free radical production could potentially sensitize doxorubicin by increasing the rate at which cleavable complexes were stabilized by doxorubicin or, alternatively, by decreasing the rate with which complexes dissociated after doxorubicin exposure. Therefore, two types of investigations were undertaken. First, the rate of stabilization of cleavable complexes was examined by exposing cells to doxorubicin for various times with or without a preceding 4 hr BSO exposure. It was determined that BSO had no effect on the rate of cleavable complex stabilization when alkaline elution studies were performed (Fig. 4). The second type of investigation was performed to assess if BSO prolonged the dissociation of stabilized cleavable complexes after doxorubicin was washed from the cells. For these experiments, BSO treatments were started 4 hr prior to the doxorubicin exposure and continued through the period allowed for dissociation of stabilized cleavable complexes. It was found that BSO did not affect the rate of dissociation of stabilized cleavable complexes after doxorubicin exposure (Fig. 5).
DISCUSSION Several observations have led to differing theories of possible mechanisms of action for the chemotherapeutic agent doxorubicin, but a unifying theory remains unclear. As mentioned in the introduction, the stabilization of cleavable complexes and the formation of free radical species by doxorubicin are both important cellular events associated with cytotoxicity in different systems. The present communication examined the interaction of these two cellular events in V79 cells. It was confirmed that BSO pre-
I. J. Radiation Oncology 0 Biology 0 Physics
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Fig. 5. Buthionine sulfoximine does not affect the rate of dissociation of cleavable complexes stabilized by doxorubicin. V79 cells were exposed to 4 hr of buthionine sulfoximine (10 mM) alone, a 1 hr coincubation with butbionine sulfoximine and doxorubicin (5 &ml) followed by various post-incubation times with buthionine sulfoximine alone. Control cells were exposed to doxorubicin alone for 1 hr followed by various post-incubation times in medium. Following the appropriate dissociation time, cells were assessed for stabilized cleavable complexes as described in
Methods and Materials. Means and standard errors of three experiments are shown. The frequency of doxorubicin-induced stabilized cleavable complexes is expressed as a percentage of the stabilized complexes present after a one hour exposure to doxorubicin. treatments (in concentrations and exposure times known to deplete GSH in V79 cells (7)) resulted in sensitization of doxorubicin cytotoxicity, suggesting that doxorubicininduced free radicals were less likely to be detoxified after GSH depletion. It was initially hypothesized that greater free radical production may increase doxorubicin sensitivity by affecting the stabilization of cleavable complexes by doxorubicin. In fact, it was determined that BSO pre-treatments did not affect the accumulation of doxorubicin into the cell, the rate of cleavable complex stabilization by doxorubicin, or the rate of dissociation of cleavable complexes. These data suggest that BSO-induced doxorubicin sensitization occurs at a step that follows the stabilization of DNA-topoisomerase II complexes or by an independent mechanism. The above data are in contrast to work performed by Gorsky and Morin (4), in which the topoisomerase II-reactive drug 4’-(9-acridinylamino)-3-methanesulfon-m-anisid-
Volume 22, Number 3, 1992
ide (m-AMSA) was examined using isolated nuclei from human HL-60 leukemia cells. They found a two-fold increase in the production of stabilized cleavable complexes by m-AMSA after the incubation of isolated nuclei with microsomes and NADPH compared to m-AMSA treatment alone. They concluded that the microsome/NADPH treatment resulted in an activated form of m-AMSA that was more proficient at stabilizing cleavable complexes. Clearly their system differs greatly from the experiments in the present study, but it is interesting to consider that whole cell preparations may prevent the type of activation seen in their study. Alternatively, it is possible that in whole cells the stabilized cleavable complex causes subsequent events which may be sensitized by increased free radical activity. The importance of cytoplasmic factors with respect to cytotoxicity from topoisomerase II-reactive drugs was recently investigated by Zwelling et al. (12) in a doxorubitin resistant human fibrosarcoma cell line (DR4) and its doxorubicin sensitive parent line (HT1080). The DR4 line was also resistant to the topoisomerase-II reactive drug etoposide compared to the parent HT1080 line and this resistance correlated with a reduction in the stabilization of DNA topoisomerase II complexes by etoposide. Additionally, there was a greater difference in the stabilization of cleavable complexes by etoposide between the DR4 cells and HT1080 line when whole cells were compared to nuclei. Further investigations revealed elevated levels of antioxidant enzymes present in the DR4 cells, compared to the HT1080 cells, suggesting that cytoplasmic factors may have a great effect on the activity of the drug prior to the stabilization of cleavable complexes. Our report, and the work of others outlined above, suggest that increased free radical activity may improve doxorubicin sensitivity. The studies of Gorsky and Morin (4) and Zwelling et al. (12) indicate that this sensitization may occur by an enhanced ability of the topoisomerase II reactive drug to stabilize cleavable complexes. In contrast, our work in V79 cells suggests that BSO-induced doxorubicin sensitization does not result from changes in the number of stabilized cleavable complexes, but rather the significance of the stabilized cleavable complex is changed. It is possible that BSO causes the cell to process the stabilized cleavable complex differently. Further work will be necessary to determine if BSO directly affects a product of the stabilized complex or whether an indirect cellular event is responsible for sensitization.
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