Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
[CANCER RESEARCH 5.1. 5237-5243. November 1. 1993] Protection from Adriamycin Cytotoxicity in LI210 Cells by Brefeldin A1 Paul J. Vichi2 and Thomas R. Tritton Vermont Cancer (.'enter ¡I*. J. V., T. K. T\ and Department of Pharmacology (T. R. T.j, College of Medicine, University of Vermont, Burlington, Vermont 05405 ABSTRACT We present studies which suggest that the cytotoxic action of Adriamy cin (ADR) may involve intracellular pathways of vesicular transport. The movement of proteins or lipids from the endoplasma- reticulum to the plasma membrane via the Golgi organelle and associated compartments exhibits several temperature-sensitive steps between 15 < and 20°C.In this same temperature range, ADR loses its cytotoxic capacity. Using the inhibitor brefeldin A (BFA), we have investigated the possible role of intravesicular trafficking in the loss of ADR activity and the induction of protection from cytotoxicity at low temperature in I 121(1 cells. We show here that cells pretreated at 37°Cfor 2 h with BFA could be protected from a subsequent exposure to ADR. The concentration causing 50% inhibition, determined by cloning in soft agar, was increased approximately 3.5 fold. 11210 cells could also be protected from the topoisomerase II inhibitors etoposide and amsacrine, but to a lesser extent; the concentration causing 50% inhibition for the latter inhibitors were increased 2-fold. Spectrofluorometric analysis of intracellular ADR accumulation revealed that there was no significant difference in the level of ADR in cells with or without BFA pretreatment. In addition, examination of ADR-induced cleavable Proteins present in the plasma membrane often require recycling and/or modification to maintain an active function. If there are indeed membrane proteins the interactions of which with distal sites are important for the cytotoxic process initiated by ADR, then these interactions should be susceptible to disruption by inhibiting intra cellular protein modification or accessibility to specific sites. It has already been demonstrated that the cytotoxicity of several topoisom erase II inhibitors can be diminished by pretreatment of cells with inhibitors of protein synthesis (3-5). However, no individual protein, other than topoisomerase II, has been unambiguously identified as being decisive in mediating the action of ADR. We summarize in this report experimental studies describing the influence of disruption of normal protein and lipid transport on the actions of ADR. MATERIALS AND METHODS Cells. Murine leukemia L12IO cells were grown in suspension in McCoy's complex formation by alkaline elution showed no significant difference in the level of DNA strand breaks in cells which had been pretreated with BFA even though there was a large difference in survival. Further exami nation of the persistence of DNA damage after a period of up to 6 h of repair revealed that cells which were pretreated with BFA removed DNA strand breaks at rates equivalent to those of cells which had received ADR directly. These results suggest that the protective effect induced by brefel din A does not involve uptake, DNA damage, or repair but instead impli cates protein or lipid interactions which may be independent of DNA damage and which may influence critical events that take place after the topoisomerase II-DNA complex has been formed. INTRODUCTION Previous work in our laboratory has investigated the reversible property of the temperature-dependent cytotoxic action of ADR.3 Below 20°C,ADR loses its ability to kill cells even though potentially toxic levels of the drug can still be accumulated within the cell (1). Once cells are raised above 20°C,however, the cytotoxic process 5A medium (GIBCO. Grand Island. NY)) containing i.-glutamine at 2 IÃŽIM and 10% heat-inactivated horse serum (GIBCO). The cells were maintained in a humidified atmosphere at 37°Cand 10% CO2 and passaged twice each week. The doubling time for L1210 cells was approximately 15 h. All experiments were performed on log phase cells (2(K1,0(X)-5(K),(HK)cells/ml). Chemicals. ADR at 20 mg/ml and m-AMSA at 9.8 /¿g/mlwere purchased from Sigma, dissolved in 70% ethanol, and maintained at -20°C. Dilutions used in cell treatments were made into 70% ethanol or McCoy's 5A medium. Brefeldin A (Sigma) at 20 mg/ml was dissolved in 100% methanol and stored at -2()°C.Taxol, supplied by the National Cancer Institute, was dissolved at 20 mg/ml in dimethyl sulfoxide, as was nocodazole (Sigma) at 1 mg/ml, and maintained at -20°C. Etoposide at 59 fig/ml was dissolved in 70% ethanol and maintained at -20°C. Cloning Assay for Cell Survival. Survival of cells after a given treatment was assessed by cloning in soft agar. The method has been published previ ously (1). Briefly, following a given treatment cells were washed three times in complete medium and resuspcnded at a concentration of lOO.(MK)cells/ml. These cell suspensions were then serially diluted 1:20 (10 ml), followed by 1:10 (3 ml) into complete McCoy's 5A medium. The entire 1:10 dilution was added to 27 ml of "cloning medium" containing McCoy's 5A medium with a final concentration of 15% horse serum, 2 ITIMi -glutamine and 0.42% Difco ensues. Concomitant with the loss of activity is the inability to detect DNA damage in cells treated with ADR at temperatures below 20°C Bacto-Agar. The cell suspension was aliquoted into five 60-mm tissue culture (2), suggesting a requirement for DNA damage leading to drug-in duced cytotoxicity. These studies also revealed that the protective process is unique to whole cells and may involve proteins of extranuclear origin since isolated nuclei accumulated ADR-induced DNA strand breaks in a dose-dependent fashion at low temperature. Fur thermore, we have shown that the cytotoxic process can be inhibited in whole cells by sequestration of extracellular drug with high mo lecular weight DNA ( 1). The induction of complete cellular protection in the presence of cytotoxic concentrations of intracellular drug sup ports the role of cell surface interactions as an important component in the initiation of cell death by ADR. Received 5/20/93; accepted 8/26/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked iitlvt-ni^t-nicnt in accordance with 18 U.S.C. Section 1734 solely lo indicate this fact. 1Supported by National Cancer Institute Grants CA44729 and CA56Ãœ40. : To whom requests for reprints should be addressed. ' The abbreviations used are: ADR. Adriamycin; m-AMSA, amsacrine; BFA. brefeldin A; ER, endoplasmic reticulum; GRP. glucose-regulated protein; PBS. phosphate-buffered saline; TON. /ran.v-Golgi network. dishes/condition (Corning), 6 ml/dish, and allowed to gel at room temperature. The 1:20 dilution for each condition was counted directly using a Coulter Counter to determine the number of cells plated/condition. Culture dishes were grown for 10-12 days in a humidified incubator at 37°Cand 10% CO2. Colonies were counted, and the average cloning efficiencies were determined for each condition. Survival was calculated by comparison to untreated con trols. Uptake of ADR. ADR was quantitated by fluorescence spectroscopy using an SLM 4800 instrument. Approximately 2.5-3.5 X 10s log phase L1210 cells were concentrated to 2-3 X 10'' cells/ml and treated with or without BFA (10 /J.M)for 2 h at 37°C.Initial treatments were then divided into equal aliquots (10 ml) and the cells were treated ±ADR for 2 h at 37°C.Cells were centrifugcd at 500 x g for 5 min at 4°C,and the cell pellets were washed 3 times in ice-cold PBS (pH 7.4). The cell pellet was then resuspended in 2 ml of Na2CO.ì (pH 9.0), and the lysate was transferred to glass 18 mm x 150 mm test tubes. ADR was extracted from the lysate by three successive additions (3 ml each) of an organic buffer containing ethyl acetate and /i-propyl alcohol in a ratio of 9:1, respectively. Tubes were vortexed vigorously and then centrifugcd at 1000 X g for 15 min at 4°Cto efficiently separate the organic layer from the water layer. The organic layer was removed and pooled for each condition. These were then dried under N2 and the residues were resuspended in 2 ml of ethanol at 70%. ADR content for each condition was determined by fluores- 5237 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research. PROTECTION FROM ADRIAMYC1N BY BREFELDIN A cencc and comparison to known standards. Excitation was at 470 nm and the emission was collected using a 550 nm long-pass filter. reduced by disruption of vesicular movement. To test this hypothesis, L1210 cells were pretreated for 2 h at 37°Cwith 10 /LIMBFA. The Alkaline Elution. Total DNA strand breaks were assayed using alkaline elution as described elsewhere (6). Forty ml of L1210 cells at 5 X IO4 cells/ml in complete McCoy's 5A medium were labeled with either 0.1 ^iCi/ml of [14C]thymidinc (ICN) or O.I ¿iCi/mlof pHlthymidine (ICN) for 24 h at 37°C fungal metabolite BFA has been reported to reversibly disrupt anterograde vesicular trafficking between the ER and the Golgi apparatus (7) by inducing the dissociation of a Mr 110,000 protein critical for vesicular budding and fusion from the membrane of the Golgi organelle (8). After pretreatment with BFA, the cells were subsequently exposed to various concentrations of ADR for 2 more h at 37°Cin the and 10% COi. The cells were then centrifuged and resuspended in 50 ml of complete McCoy's 5A medium without radionucleotide and chased for ap proximately 18 h. The cells labeled with [l4C]thymidine were then counted using a hemacytometer and treatments were performed. Following all treat ments, the cells were washed with ice-cold PBS-6 rtiM EDTA (pH 7.4) and resuspended at 1 X 10'' cells/ml in cold PBS-6 HIM EDTA. An aliquot of untreated 14C'-labeled cells and a separate aliquot of BBS-washed, 3H-labeled cells were exposed to 4(H) rads of ionizing radiation using a ^Co or 137Ce source. The irradiated cells labeled with 14C served as a calibration curve for each experiment, while the irradiated 3H-labeled cells served as an internal standard for each condition. 5.0 X IO5 14C-labeled cells, with or without treatment, and 5.0 X IO5 3H-labeled cells exposed to 400 rads were added onto 2.0 p.m polycarbonate filters. The cells were washed on the filters with 5 ml of cold PBS-6 m.MEDTA and then lysed for l h at room temperature with lysis buffer containing 2% sodium dodecyl sulfate, 30 ITIMdisodium EDTA, and 0.5 mg/ml proteinase K (pH 10. l ). Lysates were washed with 5 ml 20 HIMdisodium EDTA (pH 10.1). The DNA was then eluted overnight with 20 ml of elution buffer containing 0.1% sodium dodecyl sulfate, 2.0% tetrapropylammonium hydroxide, and 20 ITIMEDTA (free acid; pH 12.2). Eight 2.5-ml fractions were presence of BFA and cell survival was determined by cloning in soft agar. As shown in Fig. IA, the percentage survival was significantly increased if cells were pretreated with BFA prior to an exposure to ADR. The concentration causing 50% inhibition was increased from -0.03 /XMto 0.1 /AM. The protection that cells acquired from ADR cytotoxicity was par tially dependent on the concentration of BFA at which cells were pretreated. Fig. IB indicates that the cell survival increased in cells pretreated for 1 h with increasing concentrations of BFA prior to a 2-h exposure to 0.1 JAMADR. Maximal protection was attained by pre treatment with 100 JAMBFA, although the effect was not dramatically increased over lower concentrations (10 /XM).BFA by itself was not toxic at the concentrations and times used. It is important to note, however, that although low temperature can provide complete protec- collected/condition. Twelve ml of Ecolume (ICN) were added to each fraction and the I4C and 'H content was quantitated on a Packard 2500TR liquid scintillation counter. The polycarbonate filters from each condition were dis solved in 750 ft\ of Protosol (NEN) and the isotope content was quantitated by addition of 5 ml of Ecolume and liquid scintillation counting. The data were graphed as the percentage of [3H]thymidine retained on the filter versus the percentage of [l4C]thymidine retained on the filter. Calculations for singlestrand breaks (SSB) were quantitated using 14C values obtained for each condition when 40% of the internal standard ('H + 400 rads) remained on the Õ 3 M filter. SSB = log(Ireated/untrcalctl) log(X-ray/untreated) The results were expressed in rad equivalents by multiplying the frequency of single-strand breaks by the dose of radiation, in rads, of the calibration 1.00 0.10 curve. For all experiments, this was 400 rads. Repair of DNA Damage. The extent of DNA breaks remaining after 3 or 6 h of repair was assayed using the alkaline elution technique described above with the following modifications. After treatment of '4C-labeled cells with or ADR without 10 /AMBFA and 0.14 /AMADR, the cells were washed 3 times in chilled PBS with or without 10 /AMBFA. The cells were then resuspended in complete McCoy's 5A medium with 10% horse serum and allowed to repair at 37°Cfor up to 6 h. The cells were then centrifuged and resuspended at 1 X IO6 cells/ml in PBS-6 mm EDTA and layered on 2.0 /Ampolycarbonate filters (Nuclcopore). The cells were washed and lysed, and the DNA was eluted as described in "Alkaline Elution." The calibration line for repair experiments was created using 14C- and 'H-labeled cells which had been exposed to 450 rads of ionizing radiation from a '37Ce source. tn M Measurement of DNA Synthesis. Log phase L1210 cells were centrifuged and resuspended at 1 x K)1'cells/ml in complete McCoy's 5A medium with L-glutamine and 10% horse serum. BFA (10 /AM)was added and the cells were aliquoted into %-well plates at 100 /Al/well. The cells were incubated for 2 h at 37°Cbefore addition of [3H]thymidine at 1 /ACi/well. The cells were incu bated for an additional 2 h at 37°C,and the level of incorporation of [3H]- 5E-1 1 100 Brefeldin A ¿ÌM thymidine was determined at various intervals. Cells were harvested onto glass fiber filters and the content of ['Hjthymidine was determined using a Packard Fig. 1. (A) Effect of brefeldin A pretreatment on Adriamycin-induced cytotoxicity. L1210 cells at 2-4 X IO5 cells/ml were pretrealed with K) /J.MBFA for 2 h at 37°C.The Matrix 96. cells were then exposed to various concentrations of Adriamycin for an additional 2 h at 37°Cand washed, and the cell survival was measured by cloning in soft agar. Data RESULTS Effect of BFA on ADR-induced Cytotoxicity. If protein transport processes contribute to ADR-induced cytotoxicity, then the degree of cytotoxicity induced by a given concentration of ADR should be represent the summation of 5 independent experiments with 5 replicates/data point for each experiment. (B) Influence of increasing concentrations of brefeldin A during pre treatment on the protection from ADR cytotoxicity. L1210 cells were pretreated with various concentrations of BFA for 1 h at 37°C.The cells were then exposed to 0. l /J.MADR for 2 h at 37°Cand washed; the percentage survival was determined by cloning in soft agar. The average survival of nonpretreated cells was 8.3%. Data represent the mean of 3 independent experiments each with 5 replicates/data point. Bars, SD. 5238 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research. PROTECTION FROM ADRIAMYCIN tion from any concentration of ADR, the protection provided by BFA was not absolute and could be completely overwhelmed at ADR concentrations >0.25 JXM. We also examined the effect of BFA on drug-induced cytotoxicity of the additional topoisomerase II inhibitors etoposide and m-AMSA. BY BREFELDIN A 100 O - nocodazole •¿ + nocodazole Fig. 2 indicates that cells could also be protected from etoposide and m-AMSA by preincubation with 10 /J.MBFA for 2 h. The concentra tion causing 50% inhibition for both agents was increased approxi mately 2-fold. This protection was not as exaggerated as that observed for ADR, however, and may be a reflection of the manner with which each of these agents interacts with the cell. Influence of Nocodazole and Taxol on ADR Cytotoxicity. In order to test if microtubule-based transport is involved in the induction of ADR cytotoxicity, L1210 cells were pretreated with 5 /J.Mnocodazole, an inhibitor of microtubule polymerization (9), for l h at 37°C before exposure to ADR for an additional 2 h at 37°C.Nocodazole 0.000 0.025 0.050 0.075 ADR MM was also present during exposure to ADR. Survival was assayed by cloning in soft agar. The results illustrated in Fig. 3^4 show that pretreated cells had an increased survival of approximately 2-fold when subsequently exposed to ADR. Although the disruption of mi crotubule formation can result in the inhibition of other cellular pro cesses in addition to protein transport, most notably mitosis, there is no evidence to suggest that such inhibition would lead to protection 120100 D n en M ADR MM Fig. 3. (A) Effect of microtubule disruption at 37°Cby nocodazole on ADR- induced cytotoxicity. L1210 cells were pretreated with 5 (ÃŒM nocodazole for l h al 37°C.The cells were subsequently exposed to various concentrations of Adriamycin for 2 h at 37°Cand washed; survival was determined by cloning in soft agar. Data represent the mean of 3 independent experiments each with 5 replicates/data point. Bars. SD. (B) Stabilization of microtubules at low temperature by taxol and the effect on ADR cytotoxicity. L1210 cells were exposed to taxol (40 JUM)for 30 min at 37°C.The cells were transferred to ()°Cfor 2 h, exposed to ADR for an additional 2 h at ()°C,washed, and cloned in soft agar. Data 0.6 0.0 represent the mean of 3 independent experiments with 5 replicates/data point. Bars, SD. 120» O—O 1000 0.000 from ADR-induced cell death. ADR has, by itself, been shown to cause a blockade of cells in the G2-M phase of the cell cycle (10-13). Stabilization of microtubules has been demonstrated to be an im portant component of the activity of the antitumor agent taxol (1416). At low temperature, cytoplasmic microtubules have been shown to be unstable, losing their filamentous organization (17, 18). This can be prevented in whole cells by pretreatment with taxol (14, 16, 19). These stabilized microtubules are resistant to depolymerization in duced by low temperature as well as drugs. We hypothesized that if destabilization of microtubules contributed toward the protection from ADR which cells acquired below 20°C,then perhaps by maintaining - BFA •¿â€”• + BFA 0.025 0.050 0.075 0.100 m-AMSA MM Fig. 2. (A) Effect of brefeldin A pretreatment on the cytotoxicity of eloposide (V7>/6). L12IO cells were pretreated at 37°Cwith 10 ^LMBFA for 2 h. The cells were subsequently exposed to different concentrations of etoposide for 2 additional h at 37°C,washed, and cloned in soft agar. Data represent the mean of 3 independent experiments each with 5 replicates/data point. Bars. SD. (B) Effect of BFA pretreatment on the cytotoxicity induced by m-AMSA. Cells were pretreated with IO U.MBFA for 2 h at 37°Cand then exposed to various concentrations of m-AMSA for an additional 2 h at 37°C.The percentage survival was determined by cloning in soft agar. Data represent the mean of independent experiments each with 5 replicates/data point. Bars, SD. microtubules at low temperature we might overcome the protective process. We therefore attempted to stabilize microtubules by addition of taxol prior to an exposure to ADR. Fig. 35 illustrates that pretreat ment with 40 JUMtaxol did not significantly alter the protection cells acquired from ADR at 0°C.L1210 cells were pretreated with taxol for 30 min at 37°Cto facilitate uptake and interactions before shifting to 0°Cfor an additional 2 h. At this point, the cells were exposed to ADR for 2 h at 0°Cand their survival was determined. Virtually all cells survived an exposure to 1 JU.M,2 /J.M,or 5 JIMADR with or without taxol pretreatment. Although taxol has been shown to maintain mi 3 crotubules in platelets at low temperature, these structures differed 5239 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research. PROTECTION FROM ADR1AMYCIN A with DNA. Using alkaline elution, we examined the amount of total ADR-induced single-stranded DNA breaks in cells which had been pretreated with 10 /J.MBFA for 1 h, prior to a 2-h exposure to various concentrations of ADR. At all ADR concentrations examined, there were no significant differences in the formation of strand breaks in BFA-pretreated cells compared to cells receiving ADR directly (Fig. 5). Persistence of ADR-induced DNA Damage. The ability of BFApretreated cells to accumulate levels of DNA damage similar to those of nonpretreated cells suggests that involvement of interactions which may occur independent of DNA damage, or subsequent to the forma tion of the cleavable complex, may have a critical role in whether the cell dies. The persistence of DNA damage is one aspect which we thought warranted investigation. It is possible, for example, that DNA damage accumulated under BFA may be more readily removed or repaired, thereby not contributing as significantly toward the cytotoxic process. We therefore measured the levels of DNA strand breaks which remained after a period of repair. The results presented in Fig. 6 show that after 3 or 6 h of repair, equivalent percentages of initial DNA breaks remained in cells which had been pretreated with BFA compared to cells treated directly with ADR. Role of DNA Synthesis in Protection by BFA and Cytotoxicity Induced by ADR. An additional area deserving investigation was the effect that BFA may have on the ability of the cell to maintain DNA synthesis. Other laboratories have shown that the cytotoxicity of the topoisomerase agents m-AMSA and etoposide could be reduced if cells were pretreated with agents which inhibited nucleotide synthesis (20, 21). In order to test the possibility that BFA may be inducing protection via an alteration of the rate of DNA synthesis, L1210 cells were pretreated for 2 h at 37°Cwith 10 /MMBFA and then allowed to incorporate [3H]thymidine for an additional 2 h at 37°Cin the pres 100 10 ADR O.tuM BY BREFELD1N ADR 1.0uM Fig. 4. Effect of BFA prctrcatment on the uptake of ADR. Log phase cells were concentrated and pretreated with 10 U,MBFA for 2 h at 37°C.The cells were subsequently incubated with either 0.1 /¿M or 1.0 ¡ÕM ADR for 2 h at 37°Cand washed. Adriamycin was ence of BFA. The results illustrated in Fig. 7 show that BFA did not reduce the ability of L1210 cells to incorporate labeled nucleotide. extracted and the content was quantitated by fluorescent analysis. Data are expressed in pmol of Adriamycin accumulated/10'1 cells as a function of treatment. In each experiment the conditions were assayed in duplicate. Data represent the mean of 3 independent experiments. Bars, SE. DISCUSSION ADR is a topoisomerase II inhibitor which has enjoyed consider able success in the clinic despite the lack of a full understanding of its mechanism of action. While the drug has been shown to cause DNA damage via stabilization of the topoisomerase II cleavage complex (22), there is incomplete information describing various interactions which may regulate the ability of ADR to stabilize the topoisomerase significantly from platelets which were observed at 37°C(19). At low temperature, microtubules were not organized into the circumferential bands normally present in platelets but were seen more as masses or bundles within the cell. Therefore, taxol may preserve or allow mi crotubules to exist at low temperature but may not be sufficient to maintain the preexisting architecture. Additionally, the stabilization of microtubules by taxol may be insufficient to maintain normal trans port between organelles if other processes such as fusion or budding are inhibited by low temperature. Effect of BFA on Drug Uptake. The protection from ADR ac quired by cells pretreated with BFA could be explained if BFA altered the level of ADR uptake. We therefore measured the steady-state levels of ADR accumulated in cells with or without BFA pretreatment. Fig. 4 shows that although there is a small change in the levels of ADR accumulated at 0.1 /AM(92% ±13% of nonpretreated cells), this does not appear to be sufficient to account for the greater than 3-fold difference seen in survival. At higher doses of ADR such as 1 /XM, there was a greater difference observed in total levels of intracellular drug accumulated in pretreated cells; however, BFA does not protect the cells against a drug insult of this magnitude. At this time we cannot explain why BFA seems to effect ADR accumulation at higher con centrations. Effect of BFA on Cleavable Complex Formation. Although there appear to be equivalent levels of ADR in BFA-pretreated and control cells, it is possible that the effect of pretreatment is to cause a parti tioning of the drug into specific cellular compartments, thereby re ducing the amount of ADR which reaches the nucleus and can interact 300 250200- SO & 150- m 100- t 50- -o O—O - BFA •¿ •¿ + BFA -50 0.00 0.05 0.10 0.15 0.20 ADR /¿M Fig. 5. Influence of BFA pretreatment on the level of topoisomerase-mediated DNA damage induced by ADR. LI210 cells, which had been labeled with [MC]thymidine, were exposed to 10 ^M BFA for l h at 37°C.The cells were then exposed to various concen trations of ADR for 2 h at 37°Cand washed; the level of total DNA strand breaks were measured using alkaline elution. Data represent the mean of 3 independent experiments. Bars, SE. 5240 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research. PROTECTION FROM ADRIAMYC1N 100 m •¿D I 5 •¿s M Time of Repair (hours) Fig. 6. Reversibility of ADR-induced DNA damage in cells with or without brefcldin A prctrcatment. Log phase cells labeled with [14C]thymidine were pretreated for l h at 37°Cwith H) /IM BFA before exposure to 0.14 ¿IM ADR for 2 h at 37°C.Cells were washed 3 times in PBS with or without 10 /J.MBFA and resuspended at 1 X IO5 cells/ml in complete McCoy's 5A medium at 37°C.The cells were allowed to repair for either 3 or 6 h and the levels of strand breaks remaining under each condition were assayed by alkaline etution. Results represent the mean of 4 independent experiments. Bars, SE. II-DNA complex as well as information illustrating events which take place after the DNA has been compromised. When considering the many different actions of ADR in addition to the ability to damage DNA (23-25), it has been difficult to assert a clear relationship be tween the level of drug-induced topoisomerase-mediated lesions and cell death. It has been suggested that strand separation in the presence of a stabilized, covalently bound topoisomerase II-drug complex could lead to erroneous reassociation of topoisomerase II subunits and nucleotide tails generating nonhomologous recombination events and subsequent alterations in the DNA sequence (20). In accordance, it has been demonstrated that the cytotoxicity of m-AMSA and etoposide could be diminished in cells by pretreatment with inhibitors of nucleo tide synthesis such as aphidicolin and cordycepin (20, 21). However, the protective effect initiated by inhibitors of nucleotide synthesis has not been demonstrated for ADR. Furthermore, the protection that cells acquired by pretreatment with BFA was not the result of inhibition of nucleotide incorporation into DNA. Treatment of L1210 cells with 10 /U.MBFA did not inhibit DNA synthesis. Other laboratories have shown that inhibition of protein synthesis (3-5) or depletion of intracellular calcium (26) prior to exposure to topoisomerase II inhibitors results in similar events as we have described here. In each case, the reduction in protein synthesis or calcium levels was not accompanied by a diminution in the formation of drug-induced cleavable complexes. Intravesicular trafficking of proteins to the plasma membrane, or from the plasma membrane to the interior, involves several steps, many of which display a tightly regulated temperature dependence (27). At temperatures below 20°C,internalization and degradation BY BREFELDIN A We used BFA and nocodazole in an attempt to disrupt cellular transport mechanisms and measure the influence of such disturbances on the ability of ADR to induce cell death. The drug BFA has been shown to be extremely useful as a potent, reversible agent for disrup tion of vesicular transport processes (7, 33, 34). BFA induces a rapid redistribution of the coat protein ß-COPfrom the membranes of the Golgi (8) resulting in the fusion of Golgi compartments, mixing of the Golgi into the ER, and the subsequent inhibition of anterograde move ment of proteins from the ER (33). Our rationale for using nocodazole derives from past research which has supported a role for microtubules as an important compo nent in several membrane trafficking steps within the cell (35, 36). In addition to helping maintain the organization of the Golgi apparatus, microtubules have been shown to be involved in several stages of protein movement, such as the transport of large endocytic vesicles toward the nuclear region (37) as well as the movement of vesicles between the ER and an intermediate or salvage compartment prior to the Golgi (38, 39). The organization and kinetics of microtubules have been shown to be dependent upon temperature. Observation of as sembly kinetics for in vitro tubulin preparations revealed a switch from biphasic to monophasic if the temperature was decreased below 30°C(18). Immunocytochemical examination of frog oocytes cooled to 25°C,18°C,or 4°Cindicated a progressive disruption or fragmen tation of the spindle and displacement of chromosomes (40). A 2-h low-temperature treatment at 0°Cto 4°Cwas sufficient to disrupt all but kinetochore microtubules in mitotic ptKl cells (41). We therefore reasoned that disruption of microtubule assembly and/or Golgi mor phology by low temperature may result in the partial loss of critical architecture, microtubule-based vesicular transport, and Golgi func tion in L1210 cells; these losses may contribute toward the protection from ADR acquired at low temperature. The fact that BFA provided a consistently higher degree of protection from ADR than nocodazole may be related to the degree to which each of these agents disrupts various transport processes. For example, microtubules do not appear to be involved in transporting newly synthesized proteins between the salvage compartment, Golgi cisternae or fra/w-Golgi (35, 37). Fur thermore, Golgi units which had been disrupted with nocodazole were shown to retain cis-trans polarity and functionality (42). Therefore, although the addition of nocodazole to cells causes the depolymerization of microtubules, the resulting influence on transport may be limited to specific interactions such as fusion between early and late endosomes (37), disassembly of the Golgi stacks (36), formation of rates of membrane proteins display a marked decline in activity (2729). The exit of proteins from the TON to the cell surface is also significantly reduced below 20°C(30, 31). At still lower temperatures, •¿such as 15°C,vesicles have been shown to accumulate in an inter mediate compartment between the ER and Golgi (27, 31). Stereological volume measurements of the Golgi compartment also indicate an increase in the size of the TON at 2()°Cand a decrease in the surface 90 120 area and volume of preceding Golgi compartments (32). Although Time (minutes) ADR has no identified receptor and diffuses freely into the cell at both Fig. 7. Effect of BFA on DNA synthesis. Log phase cells were concentrated to 1 X IO6 cells/ml and exposed to BFA (10 JIM) at 37°Cfor 2 h in 96-well plates (100 »¿I/well). high and low temperature, albeit with reduced kinetics at low tem [•'HjThymidincwas added at 1 /iCi/well and the cells were incubated for 2 h at 37°C. perature, there may be important regulatory proteins or cofactors Incorporation of ['H]thymidine was assayed at 30- or 60-min intervals. The results which are important in mediating cytotoxicity, yet with modification represent the mean of an individual experiment with 12 replicates/data point. Bars, SD. or transport that is prevented by low temperature. These results were repeated in 3 additional experiments. 5241 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research. PROTECTION FROM ADRIAMYCIN the TGN-early endosóme (43), movement of newly synthesized pro teins from the ER to the Golgi or from the Golgi to the plasma membrane, and recycling of receptors (34). As a result, microtubuledisrupting agents such as nocodazole may not universally inhibit the flow of membrane traffic but have instead been shown to result in the misdirection of vesicle movement (35). This lack of direction could prevent a signaling protein from reaching its intended destination or from being recycled to specific sites such as the plasma membrane or ER. Such alterations in vesicle movement may be sufficient to account for the limited protection from ADR observed in cells pretreated with nocodazole. Protection from cell death by pretreatment with BFA is most likely related to the ability of BFA to inhibit some transport process rather than an unidentified property of the drug, although this latter expla nation cannot be ruled out. The fact that temperature disrupts some of the same processes as BFA may also be coincidental; however, both conditions lead to protection from ADR-induced cytotoxicity. Tem peratures around 20°Cbegin to selectively inhibit various steps within BY BREFELDIN A shown to protect cells against doxorubicin-induced cytotoxicity (53). It is possible that the disruption of Golgi by BFA results in an eleva tion of GRPs and subsequent protection from ADR similar to that previously demonstrated (53). Additionally, part of the protective re sponse induced by low temperature may be the ability to induce an elevation of GRPs. This hypothesis will provide an active area for future investigation. REFERENCES the transport pathways. Additional processes become inhibited as the temperature is lowered to 15°C.Decreasing the temperature below 20°Cresults in complete protection from ADR cytotoxicity. The pro tection provided by BFA at 37°C,although significant, is incomplete and may be related to the degree to which BFA can prevent the movement or modification of some critical protein. Low temperature presents an absolute block to protein movement while BFA causes a partial inhibition; e.g., BFA blocks anterograde transport from the ER to the Golgi but not retrograde transfer from the plasma membrane to the TON or from the Golgi to the ER. The decreased level of protection provided by BFA against etoposide and m-AMSA compared to ADR may be a reflection of the cellular system (L1210) which we have used to conduct our studies or an indication of the manner in which each of these agents interacts with other cellular components in addition to DNA. The focus of this investigation was primarily on the interactions between BFA and ADR; however, another laboratory has shown that additional cell lines can be significantly protected from etoposide as well as ADR.4 In each case, there is a protective effect induced by BFA without complemen tary reductions in the formation of DNA cleavage complexes. Taken together, these results suggest that the cytotoxic actions of each in hibitor share a common mechanistic basis beyond inhibition of topoisomerase II, which is responsible for commitment to cell death. Al though these agents can be classified as belonging to the multidrugresistant family, at this time we do not know if BFA has a similar effect on cells which exhibit resistance to these drugs. It is interesting to note that several labs have shown that resistant cells accumulate multidrugresistant compounds in cytoplasmic vesicles (44-46). If these vesicles are being actively transported to the extracellular environment in order to facilitate decreased intracellular drug levels, then inhibition of vesicular transport by pretreatment with BFA could lead to increased intracellular drug levels and subsequent enhancement of cytotoxicity. BFA has been reported previously to protect cells from the cyto toxic effects of ricin, modeccin, and Pseudomonas toxin (47). How ever, unlike ADR, these agents enter the cell primarily via endocytosis and their active moieties are subsequently translocated from an intra cellular compartment to the cytosol (48, 49). Several recent publications are of particular interest with respect to the actions of BFA. Agents which have been shown to lead to dis ruptions in ER function (BFA; thapsigargin) have been shown to induce a family of stress proteins referred to as GRP (50-52). Induc tion of these proteins in Chinese hamster ovary cells by glucose deprivation, 2-deoxyglucose, and the calcium ionophore A23187 were 4 K. A. Kennedy, unpublished observations. Vichi, P., and Trimm. T. R. Adriamycin: protection from cell death by removal of extracellular drug. Cancer Res., 52: 4135^1138, 1992. Vichi, P., Robison. S.. and Tritton. T. R. Temperature dependence of Adriamycininduced DNA damage in L121Ãœcells. Cancer Res., 49: 5575-5580, 1989. Chow, K. C, King, C. K., and Ross. W. E. Abrogation of eloposidc-mediated cyto toxicity by cycloheximide. Biochcm. Pharmacol., 37: 1117-1122, 1988. Schneider, E., Lawson, P. A., and Ralph, R. K. Inhibition of protein synthesis reduces the cytotoxicity of 4'-(9-acridinylamino)methane-sulfon-m-aniside without affecting DNA breakage and DNA topoisomerase II in a murine mastocytoma cell line. Biochem. Pharmacol., 38: 263-269, 1989. Bonner. J. A., and Lawrence. T. S. Protection of doxorubicin cytotoxicity by cyclo heximide. Int. J. Oncol. Biol. Phys., 16: 1209-1212, 1989. Kohn. K. W., Ewig, R. A. G., Erickson, L. C., and Zwelling, L. A. Measurement of strand breaks and crosslinks by alkaline elution. In: E. C. Friedberg and P. C. Hanawolt (cds.), DNA Repair: A Laboratory Manual of Research Procedures, pp. 373-401. New York: Marcel Dekker, 1981. Misumi, Y. Mr.,nui. Y, Miki, K., Takatsuki, A., Tamura, G., and Ikehara, Y. Novel blockade by brefeldin A of intracellular transport of secretory proteins in cultured rat hepatocytes. J. Biol. Chem., 261: 11398-11403, 1986. Donaldson, J. G., Lippincott-Schwartz. J., Bloom. G. S., Kreis, T. E., Klausner, R. D. Dissociation of a 110-kD peripheral membrane protein from the Golgi apparatus is an early event in brefeldin A action. J. Cell Biol., ///: 2295-2306, 1990. Lee, J. C., Field, D. J., and Lee, L. L. Y. Effects of nocodazole on structures of calf brain tubulin. Biochemistry, 19: 6209-6215, 1980. Nakata, B., Chung, Y. S.. Yokomatsu. H.. Sawada, T., Kubo, T., Kondo, Y., Satake, K.. and Sowa, M. Flow cytometric bromodeoxyuridine/DNA analysis of hyperthermia and/or Adriamycin from human pancreatic adcnocarcinoma cell line Capan-2. Jpn. J. Cancer Res., 83: 477-482, 1992. Zucker, R. M.. Adams. D. J.. Bair, K. W., and Elstein, K. H. Polyploidy induction as a consequence of topoisomerase inhibition: a flow cytometric assessment. Biochem. Pharmacol., 42: 2199-2208, 1991. Krishan. A., and Frei, E., III. Effect of Adriamycin on the cell cycle transverse and kinetics of cultured human lymphocytes. Cancer Res., 36: 143-150. 1976. Barlogie. B., Drewinko, B., Johnston, D. A., and Freireich, E. J. The effect of Adriamycin on the cell cycle transverse of a human lymphoid cell line. Cancer Res., 36: 1975-1979, 1976. Horwitz, S. B. Mechanism of action of taxol. Trends Pharmacol. Sci., 13: 134—136, 1992. Rowinsky, E. K., Cazenave, L. A., and Donehowcr, R. C. Taxol: a novel investigational anti-microlubule agent. J. Nail. Cancer Inst., 82: 1247-1259, 1990. Schiff. P. B.. and Horwitz, S. B. Taxol stabilizes microtubules in mouse fibroblast cells. Proc. Nati. Acad. Sci. USA, 77: 1561-1565, 1980. Pirollet, F., Margolis, R. L., and Job, D. Ca2f-calmodulin regulated effectors of microtubule stability in neuronal tissues. Biochim. Biophys. Acta, 1160: 113-119, 1992. Barton, J. S., Vandivort, D. L., Heacock, D. H., Coffman, J. A., and Trygg, K. A. Microtubule assembly kinetics: changes with solution conditions. Biochem. J., 247: 505-511, 1987. White. J. G. Influence of taxol on the response of platelets to chilling. Am. J. Pathol.. 108: 184-195, 1982. Kaufmann. S. H. Antagonism between camptothecin and topoisomerase H-directed chemotherapeutic agents in a human leukemia cell line. Cancer Res., 57: 1129-1136, 1991. D'Arpa, P.. Beardmore, C., and Liu, L. F. Involvement of nucleic acid synthesis in cell killing mechanisms of topoisomerase poisons. Cancer Res., 50: 6919-6924, 1990. Liu, L. F. DNA topoisomerase poisons as antitumor drugs. Annu. Rev. Biochem., 58: 351-375, 1989. Arcamone, F. Doxorubicin: Anticancer Antibiotics, pp. 93-157. New York: Academic Press, 1981. Gianni. L., Corden, B. J., and Myers, C. E. The biochemical basis of anthracycline toxicity and antitumor activity. Rev. Biochem. Toxicol., 5: 1-82, 1983. Tritton, T. R. Cell surface actions of Adriamycin. Pharmacol. Ther., 49: 293-309, 1991. Bertrand, R., Kerrigan, D., Sarang, M., and Pommier, Y. Cell death induced by topoisomerase inhibitors. Biochem. Pharmacol., 42: 77-85, 1991. Kuismanen, E.. and Saraste, J. Low temperature-induced transport blocks as tools to manipulate membrane traffic. Methods Cell Biol., 32: 257-274, 1989. Tomoda, H.. Kishimoto, Y. and Lee, Y. C. Temperature effect on endocytosis and exocytosis by rabbit alveolar macrophages. J. Biol. Chem., 264: 15445-15450, 1989. Marsh. M., Bolzau, E., and Helenius, A. Penetration of Semliki Forest virus from acidic prelysosomal vacuoles. Cell, 32: 931-940, 1983. Rothman. J. E.. and Orci. C. Molecular dissection of the secretory pathway. Nature (Lond.), 355: 409-415, 1992. 5242 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research. PROTECTION FROM ADRIAMYCIN 31. Saraste, J., and Kuismanen, E. Pre- and posi Golgi vacuoles operale in the transport of Semliki Forest virus membrane glycoproteins to the cell surface. Cell, 38: 535549, 1984. 32. Griffiths, G., Fuller, S. D., Back, R.. Hollinshead, M.. Pfeiffer, S., and Simons, K. The dynamic nature of the Golgi complex. J. Cell Biol., 108: 277-297, 1989. 33. Klausner, R. D., Donaldson, i. G., and Lippincott-Schwartz, J. Brefeldin A: insights into the control of membrane traffic and organelle structure. J. Cell Biol., 776; 1071-1080, 1992. 34. Pelham, H. R. B. Multiple targets for brefeldin A. Cell, 67: 449-451, 1991. 35. Kelly, R. B. Microlubules, membrane traffic, and cell organization. Cell, 61: 5-7, 1990. 36. Thyberg, J., and Moskalewski, S. Microtubules and the organization of the Golgi complex. Exp. Cell Res., 159: 1-16, 1985. 37. Bruenberg. J.. Griffiths, G., and Howell, K. E. Characterization of the early endosóme and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion m viti».¡.Cell Biol., 70«:1301-1316, 1989. 38. Pelham. H. R. B. Control of protein exit from the cndoplasmic rcticulum. Annu. Rev. Cell Biol., 5: 1-23. 1989. 39. Lippincott-Schwartz. J.. Donaldson, J. G., Schweizer, A., Berger, E. G., Mauri, H. P., Yuan, L. C„and Klausner, R. D. Microtubule-dcpcndent retrograde transport of proteins into the ER in the presence of brefeldin A suggest an ER recycling pathway. Cell, 60: 821-836, 1990. 40. Pickering. S. J., and Johnson, M. H. The influence of cooling on the organization of the mciotic spindle of the mouse oocyte. Hum. Reprod. (Oxf.), 2: 207-216, 1987. 41. Sweet, S. C, and Welsh, M. J. Calmodulin colocalization with cold-stable and nocodazolc-stable microtubules in living PtKl cells. Eur. J. Cell Biol., 47: 88-93, 1988. 42. lida, H.. and Shibota, Y. Functional Golgi units in microtubule-disrupted cultured atrial myocytes. J. Histochem. Cylochem., 39: 1349-1355, 1991. 43. Wood. S. A.. Park, J. E., and Brown. W. J. Brefeldin A causes a microtubule-mediated fusion of the trans-Golgi network and early endosomes. Cell, 67: 591-600. 1991. BY BREFELDIN A 44. Sognier, M. A., Zhang, Y, Eberle, R. L., and Belli, J. A. Characterization of Adriamycin-resistant and radiation-sensitive Chinese hamster cell lines. Biochem. Pharmacol., 44: 1859-1868, 1992. 45. Gervasoni, J. E., Jr., Fields, S. Z., Krishna, S., Baker, M. A., Rosado, M., Thuraisamy, K., Hindenburg, A. A., and Taub, R. N. Subcellular distribution of daunorubicin in P-glycoprotein-positive and -negative drug-resistant cell lines using laser-assisted confocal microscopy. Cancer Res., 57: 4955-4963, 1991. 46. Warren, L., Jardillier, J. C., and Ordentlich, P. Secretion of lysosomal enzymes by drug-sensitive and multiple drug-resistant cells. Cancer Res., 57: 1996-2001, 1991. 47. Yoshida, T., Chen, C., Zhang, M., and Wu. H. C. Disruption of the Golgi apparatus by brefeldin A inhibits the cytotoxicity of ricin, modeccin, and Pseudomonas toxin. Exp. Cell Res., 792: 389-395, 1991. 48. Sandvig, K., Prydz, K., Hansen, S. H., and van Deurs. B. Ricin transport in brefeldin A-treated cells: correlation between Golgi structure and toxic effect. J. Cell Biol.. 775: 971-981, 1991. 49. Sandvig, K., and Olsnes. S. Entry of the toxic proteins abrin, modeccin. ricin, and diphtheria toxin into cells: effect of pH, metabolic inhibitors, and ionophores and evidence for toxin penetration from endocytotic vesicles. J. Biol. Chem., 257: 75047513, 1982. 50. Liu, E. S., Ou, J-H., and La, A. S. Brefeldin A as a regulator of GRP 78 expression in mammalian cells. J. Biol. Chem., 267: 7128-7133, 1992. 51. Lee, A. S. Mammalian stress response: induction of the glucose-regulated protein family. Curr. Opin. Cell Biol., 4: 267-273, 1992. 52. Price, B. D., Mannheim-Rodmann, L. A., and Calderwood, S. K. Brefeldin A, thapsigargin, and A1F4_ stimulate the accumulation of GRP 78 mRNA in a cycloheximide dependent manner, whilst induction by hypoxia is independent of protein synthesis. J. Physiol. (Lond.), 752: 545-552, 1992. 53. Shen, J.. Hughes, C., Chao, C.. Cai, J.. Bartels. C., Gessner, T., and Subjeck, J. Conduction of glucose regulated proteins and doxorubicin resistance in Chinese hamster cells. Proc. Nati. Acad. Sci. USA, 84: 3278-3282, 1987. 5243 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research. Protection from Adriamycin Cytotoxicity in L1210 Cells by Brefeldin A Paul J. Vichi and Thomas R. Tritton Cancer Res 1993;53:5237-5243. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/53/21/5237 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research.