Protection from Adriamycin Cytotoxicity in LI210 Cells by Brefeldin A1
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[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 Proteins present in the plasma membrane often require recycling and/or modification to maintain an active function. If there are indeed We present studies which suggest that the cytotoxic action of Adriamy membrane proteins the interactions of which with distal sites are cin (ADR) may involve intracellular pathways of vesicular transport. The movement of proteins or lipids from the endoplasma- reticulum to the important for the cytotoxic process initiated by ADR, then these interactions should be susceptible to disruption by inhibiting intra plasma membrane via the Golgi organelle and associated compartments exhibits several temperature-sensitive steps between 15 < and 20°C.In cellular protein modification or accessibility to specific sites. It has this same temperature range, ADR loses its cytotoxic capacity. Using the already been demonstrated that the cytotoxicity of several topoisom inhibitor brefeldin A (BFA), we have investigated the possible role of erase II inhibitors can be diminished by pretreatment of cells with intravesicular trafficking in the loss of ADR activity and the induction of inhibitors of protein synthesis (3-5). However, no individual protein, protection from cytotoxicity at low temperature in I 121(1 cells. We show other than topoisomerase II, has been unambiguously identified as here that cells pretreated at 37°Cfor 2 h with BFA could be protected from being decisive in mediating the action of ADR. We summarize in this a subsequent exposure to ADR. The concentration causing 50% inhibition, report experimental studies describing the influence of disruption of determined by cloning in soft agar, was increased approximately 3.5 fold. normal protein and lipid transport on the actions of ADR. 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. Spectrofluo- MATERIALS AND METHODS rometric analysis of intracellular ADR accumulation revealed that there was no significant difference in the level of ADR in cells with or without Cells. Murine leukemia L12IO cells were grown in suspension in McCoy's BFA pretreatment. In addition, examination of ADR-induced cleavable 5A medium (GIBCO. Grand Island. NY)) containing i.-glutamine at 2 IÃŽIMand complex formation by alkaline elution showed no significant difference in 10% heat-inactivated horse serum (GIBCO). The cells were maintained in a the level of DNA strand breaks in cells which had been pretreated with humidified atmosphere at 37°Cand 10% CO2 and passaged twice each week. BFA even though there was a large difference in survival. Further exami The doubling time for L1210 cells was approximately 15 h. All experiments nation of the persistence of DNA damage after a period of up to 6 h of were performed on log phase cells (2(K1,0(X)-5(K),(HK)cells/ml). repair revealed that cells which were pretreated with BFA removed DNA Chemicals. ADR at 20 mg/ml and m-AMSA at 9.8 /¿g/mlwere purchased strand breaks at rates equivalent to those of cells which had received ADR from Sigma, dissolved in 70% ethanol, and maintained at -20°C. Dilutions directly. These results suggest that the protective effect induced by brefel used in cell treatments were made into 70% ethanol or McCoy's 5A medium. din A does not involve uptake, DNA damage, or repair but instead impli Brefeldin A (Sigma) at 20 mg/ml was dissolved in 100% methanol and stored cates protein or lipid interactions which may be independent of DNA at -2()°C.Taxol, supplied by the National Cancer Institute, was dissolved at 20 damage and which may influence critical events that take place after the mg/ml in dimethyl sulfoxide, as was nocodazole (Sigma) at 1 mg/ml, and topoisomerase II-DNA complex has been formed. maintained at -20°C. Etoposide at 59 fig/ml was dissolved in 70% ethanol and maintained at -20°C. INTRODUCTION 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 Previous work in our laboratory has investigated the reversible ously (1). Briefly, following a given treatment cells were washed three times property of the temperature-dependent cytotoxic action of ADR.3 in complete medium and resuspcnded at a concentration of lOO.(MK)cells/ml. Below 20°C,ADR loses its ability to kill cells even though potentially 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 toxic levels of the drug can still be accumulated within the cell (1). Once cells are raised above 20°C,however, the cytotoxic process 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 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 duced cytotoxicity. These studies also revealed that the protective Counter to determine the number of cells plated/condition. Culture dishes were process is unique to whole cells and may involve proteins of extra- grown for 10-12 days in a humidified incubator at 37°Cand 10% CO2. nuclear origin since isolated nuclei accumulated ADR-induced DNA Colonies were counted, and the average cloning efficiencies were determined strand breaks in a dose-dependent fashion at low temperature. Fur for each condition. Survival was calculated by comparison to untreated con thermore, we have shown that the cytotoxic process can be inhibited trols. in whole cells by sequestration of extracellular drug with high mo Uptake of ADR. ADR was quantitated by fluorescence spectroscopy using lecular weight DNA ( 1). The induction of complete cellular protection 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 in the presence of cytotoxic concentrations of intracellular drug sup /J.M)for 2 h at 37°C.Initial treatments were then divided into equal aliquots (10 ports the role of cell surface interactions as an important component ml) and the cells were treated ±ADR for 2 h at 37°C.Cells were centrifugcd in the initiation of cell death by ADR. 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. Received 5/20/93; accepted 8/26/93. The costs of publication of this article were defrayed in part by the payment of page ADR was extracted from the lysate by three successive additions (3 ml each) charges. This article must therefore be hereby marked iitlvt-ni^t-nicnt in accordance with of an organic buffer containing ethyl acetate and /i-propyl alcohol in a ratio 18 U.S.C. Section 1734 solely lo indicate this fact. of 9:1, respectively. Tubes were vortexed vigorously and then centrifugcd at 1Supported by National Cancer Institute Grants CA44729 and CA56Ãœ40. 1000 X g for 15 min at 4°Cto efficiently separate the organic layer from the : To whom requests for reprints should be addressed. ' The abbreviations used are: ADR. Adriamycin; m-AMSA, amsacrine; BFA. brefeldin water layer. The organic layer was removed and pooled for each condition. A; ER, endoplasmic reticulum; GRP. glucose-regulated protein; PBS. phosphate-buffered These were then dried under N2 and the residues were resuspended in 2 ml of saline; TON. /ran.v-Golgi network. ethanol at 70%. ADR content for each condition was determined by fluores- 5237 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 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 reduced by disruption of vesicular movement. To test this hypothesis, emission was collected using a 550 nm long-pass filter. L1210 cells were pretreated for 2 h at 37°Cwith 10 /LIMBFA. The Alkaline Elution. Total DNA strand breaks were assayed using alkaline fungal metabolite BFA has been reported to reversibly disrupt antero- 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 grade vesicular trafficking between the ER and the Golgi apparatus (7) [14C]thymidinc (ICN) or O.I ¿iCi/mlof pHlthymidine (ICN) for 24 h at 37°C by inducing the dissociation of a Mr 110,000 protein critical for vesicular budding and fusion from the membrane of the Golgi organ- 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 elle (8). After pretreatment with BFA, the cells were subsequently exposed to various concentrations of ADR for 2 more h at 37°Cin the proximately 18 h.