Protection from Adriamycin Cytotoxicity in LI210 Cells by Brefeldin A1

[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 Â¡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 20Â°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 37Â°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 37Â°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 /Â¿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 -20Â°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()Â°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 -20Â°C. Etoposide at 59 fig/ml was dissolved in 70% ethanol and maintained at -20Â°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 20Â°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 20Â°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 20Â°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 37Â°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 37Â°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 Â±ADR for 2 h at 37Â°C.Cells were centrifugcd in the initiation of cell death by ADR. at 500 x g for 5 min at 4Â°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.Ã¬ (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 4Â°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 37Â°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 Â¿iCi/mlof pHlthymidine (ICN) for 24 h at 37Â°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 37Â°Cin the proximately 18 h. The cells labeled with [l4C]thymidine were then counted using a hemacytometer and treatments were performed. Following all treat presence of BFA and cell survival was determined by cloning in soft ments, the cells were washed with ice-cold PBS-6 rtiM EDTA (pH 7.4) and agar. As shown in Fig. IA, the percentage survival was significantly resuspended at 1 X 10'' cells/ml in cold PBS-6 HIMEDTA. An aliquot of increased if cells were pretreated with BFA prior to an exposure to untreated 14C'-labeled cells and a separate aliquot of BBS-washed, 3H-labeled ADR. The concentration causing 50% inhibition was increased from cells were exposed to 4(H) rads of ionizing radiation using a ^Co or 137Ce -0.03 /XMto 0.1 /AM. source. The irradiated cells labeled with 14C served as a calibration curve for The protection that cells acquired from ADR cytotoxicity was par each experiment, while the irradiated 3H-labeled cells served as an internal tially dependent on the concentration of BFA at which cells were 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 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 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 exposure to 0.1 JAMADR. Maximal protection was attained by pre buffer containing 2% sodium dodecyl sulfate, 30 ITIMdisodium EDTA, and 0.5 treatment with 100 JAMBFA, although the effect was not dramatically mg/ml proteinase K (pH 10. l ). Lysates were washed with 5 ml 20 HIMdisodium increased over lower concentrations (10 /XM).BFA by itself was not EDTA (pH 10.1). The DNA was then eluted overnight with 20 ml of elution toxic at the concentrations and times used. It is important to note, buffer containing 0.1% sodium dodecyl sulfate, 2.0% tetrapropylammonium however, that although low temperature can provide complete protec- hydroxide, and 20 ITIMEDTA (free acid; pH 12.2). Eight 2.5-ml fractions were 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 single- strand breaks (SSB) were quantitated using 14C values obtained for each Ã• condition when 40% of the internal standard ('H + 400 rads) remained on the 3 M filter.

log(Ireated/untrcalctl) SSB = log(X-ray/untreated)

The results were expressed in rad equivalents by multiplying the frequency 0.10 1.00 of single-strand breaks by the dose of radiation, in rads, of the calibration curve. For all experiments, this was 400 rads. ADR 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 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 37Â°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 tn using 14C- and 'H-labeled cells which had been exposed to 450 rads of ionizing M radiation from a '37Ce source. 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 37Â°Cbefore addition of [3H]thymidine at 1 /ACi/well. The cells were incu 5E-1 1 100 bated for an additional 2 h at 37Â°C,and the level of incorporation of [3H]- Brefeldin A Â¿ÃŒM 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 37Â°C.The Matrix 96. cells were then exposed to various concentrations of Adriamycin for an additional 2 h at 37Â°Cand washed, and the cell survival was measured by cloning in soft agar. Data represent the summation of 5 independent experiments with 5 replicates/data point for RESULTS 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 37Â°C.The cells were then exposed to 0. l /J.MADR Effect of BFA on ADR-induced Cytotoxicity. If protein transport for 2 h at 37Â°Cand washed; the percentage survival was determined by cloning in soft processes contribute to ADR-induced cytotoxicity, then the degree of agar. The average survival of nonpretreated cells was 8.3%. Data represent the mean of 3 cytotoxicity induced by a given concentration of ADR should be independent experiments each with 5 replicates/data point. Bars, SD. 5238

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1993 American Association for Cancer Research. PROTECTION FROM ADRIAMYCIN BY BREFELDIN A tion from any concentration of ADR, the protection provided by BFA 100 was not absolute and could be completely overwhelmed at ADR O - nocodazole concentrations >0.25 JXM. â€¢¿+nocodazole We also examined the effect of BFA on drug-induced cytotoxicity of the additional topoisomerase II inhibitors etoposide and m-AMSA. 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 0.000 0.025 0.050 0.075 of ADR cytotoxicity, L1210 cells were pretreated with 5 /J.Mnoco- dazole, an inhibitor of microtubule polymerization (9), for l h at 37Â°C before exposure to ADR for an additional 2 h at 37Â°C.Nocodazole 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 120- when subsequently exposed to ADR. Although the disruption of mi crotubule formation can result in the inhibition of other cellular pro 100 cesses in addition to protein transport, most notably mitosis, there is no evidence to suggest that such inhibition would lead to protection

en M ADR MM Fig. 3. (A) Effect of microtubule disruption at 37Â°Cby nocodazole on ADR- induced cytotoxicity. L1210 cells were pretreated with 5 (ÃŒMnocodazolefor l h al 37Â°C.The cells were subsequently exposed to various concentrations of Adriamycin for 2 h at 37Â°Cand washed; survival was determined by cloning in soft agar. Data represent the mean of 3 0.0 0.6 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 37Â°C.The cells were transferred to ()Â°Cfor 2 h, exposed to ADR for an additional 2 h at ()Â°C,washed, and cloned in soft agar. Data represent the mean of 3 independent experiments with 5 replicates/data point. Bars, SD.

120Â» from ADR-induced cell death. ADR has, by itself, been shown to Oâ€”O - BFA cause a blockade of cells in the G2-M phase of the cell cycle (10-13). 1000 â€¢¿â€”â€¢+BFA Stabilization of microtubules has been demonstrated to be an im portant component of the activity of the antitumor agent taxol (14- 16). 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 20Â°C,then perhaps by maintaining microtubules at low temperature we might overcome the protective 0.000 0.025 0.050 0.075 0.100 process. We therefore attempted to stabilize microtubules by addition of taxol prior to an exposure to ADR. Fig. 35 illustrates that pretreat m-AMSA MM ment with 40 JUMtaxol did not significantly alter the protection cells Fig. 2. (A) Effect of brefeldin A pretreatment on the cytotoxicity of eloposide (V7>/6). acquired from ADR at 0Â°C.L1210 cells were pretreated with taxol for L12IO cells were pretreated at 37Â°Cwith 10 ^LMBFA for 2 h. The cells were subsequently 30 min at 37Â°Cto facilitate uptake and interactions before shifting to exposed to different concentrations of etoposide for 2 additional h at 37Â°C,washed, and 0Â°Cfor an additional 2 h. At this point, the cells were exposed to ADR cloned in soft agar. Data represent the mean of 3 independent experiments each with 5 for 2 h at 0Â°Cand their survival was determined. Virtually all cells 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 37Â°Cand then exposed to various concentrations of m-AMSA for an additional 2 h at 37Â°C.The survived an exposure to 1 JU.M,2/J.M,or 5 JIMADR with or without percentage survival was determined by cloning in soft agar. Data represent the mean of 3 taxol pretreatment. Although taxol has been shown to maintain mi independent experiments each with 5 replicates/data point. Bars, SD. crotubules in platelets at low temperature, these structures differed 5239

100 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 BFA- pretreated 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 10 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 ADR O.tuM ADR 1.0uM protection via an alteration of the rate of DNA synthesis, L1210 cells were pretreated for 2 h at 37Â°Cwith 10 /MMBFAand then allowed to 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 37Â°C.The cells were subsequently incorporate [3H]thymidine for an additional 2 h at 37Â°Cin the pres incubated with either 0.1 /Â¿Mor1.0 Â¡Ã•MADRfor 2 h at 37Â°Cand washed. Adriamycin was ence of BFA. The results illustrated in Fig. 7 show that BFA did not 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 reduce the ability of L1210 cells to incorporate labeled nucleotide. the conditions were assayed in duplicate. Data represent the mean of 3 independent experiments. Bars, SE. DISCUSSION significantly from platelets which were observed at 37Â°C(19). At low ADR is a topoisomerase II inhibitor which has enjoyed consider able success in the clinic despite the lack of a full understanding of its temperature, microtubules were not organized into the circumferential mechanism of action. While the drug has been shown to cause DNA bands normally present in platelets but were seen more as masses or damage via stabilization of the topoisomerase II cleavage complex bundles within the cell. Therefore, taxol may preserve or allow mi (22), there is incomplete information describing various interactions crotubules to exist at low temperature but may not be sufficient to which may regulate the ability of ADR to stabilize the topoisomerase 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 300 are inhibited by low temperature. 250- Effect of BFA on Drug Uptake. The protection from ADR ac quired by cells pretreated with BFA could be explained if BFA altered 200- the level of ADR uptake. We therefore measured the steady-state levels of ADR accumulated in cells with or without BFA pretreatment. S 150- O Fig. 4 shows that although there is a small change in the levels of ADR & accumulated at 0.1 /AM(92% Â±13% of nonpretreated cells), this does m-o 100- not appear to be sufficient to account for the greater than 3-fold t 50- Oâ€”O - BFA difference seen in survival. At higher doses of ADR such as 1 /XM, â€¢¿ â€¢¿+BFA 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 -50 explain why BFA seems to effect ADR accumulation at higher con 0.00 0.05 0.10 0.15 0.20 centrations. ADR /Â¿M Effect of BFA on Cleavable Complex Formation. Although there Fig. 5. Influence of BFA pretreatment on the level of topoisomerase-mediated DNA appear to be equivalent levels of ADR in BFA-pretreated and control damage induced by ADR. LI210 cells, which had been labeled with [MC]thymidine, were exposed to 10 ^M BFA for l h at 37Â°C.The cells were then exposed to various concen cells, it is possible that the effect of pretreatment is to cause a parti trations of ADR for 2 h at 37Â°Cand washed; the level of total DNA strand breaks were tioning of the drug into specific cellular compartments, thereby re measured using alkaline elution. Data represent the mean of 3 independent experiments. ducing the amount of ADR which reaches the nucleus and can interact Bars, SE. 5240

We used BFA and nocodazole in an attempt to disrupt cellular 100 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 ÃŸ-COPfrom the membranes of the m â€¢¿D Golgi (8) resulting in the fusion of Golgi compartments, mixing of the Golgi into the ER, and the subsequent inhibition of anterograde move

I ment of proteins from the ER (33). Our rationale for using nocodazole derives from past research 5â€¢¿s which has supported a role for microtubules as an important compo M 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 Time of Repair (hours) protein movement, such as the transport of large endocytic vesicles Fig. 6. Reversibility of ADR-induced DNA damage in cells with or without brefcldin toward the nuclear region (37) as well as the movement of vesicles A prctrcatment. Log phase cells labeled with [14C]thymidine were pretreated for l h at between the ER and an intermediate or salvage compartment prior to 37Â°Cwith H) /IM BFA before exposure to 0.14 Â¿IMADR for 2 h at 37Â°C.Cells were washed 3 times in PBS with or without 10 /J.MBFA and resuspended at 1 X IO5 cells/ml the Golgi (38, 39). The organization and kinetics of microtubules have in complete McCoy's 5A medium at 37Â°C.The cells were allowed to repair for either 3 been shown to be dependent upon temperature. Observation of as or 6 h and the levels of strand breaks remaining under each condition were assayed by sembly kinetics for in vitro tubulin preparations revealed a switch alkaline etution. Results represent the mean of 4 independent experiments. Bars, SE. from biphasic to monophasic if the temperature was decreased below 30Â°C(18). Immunocytochemical examination of frog oocytes cooled to 25Â°C,18Â°C,or 4Â°Cindicated a progressive disruption or fragmen II-DNA complex as well as information illustrating events which take tation of the spindle and displacement of chromosomes (40). A 2-h place after the DNA has been compromised. When considering the low-temperature treatment at 0Â°Cto 4Â°Cwas sufficient to disrupt all many different actions of ADR in addition to the ability to damage but kinetochore microtubules in mitotic ptKl cells (41). We therefore DNA (23-25), it has been difficult to assert a clear relationship be reasoned that disruption of microtubule assembly and/or Golgi mor tween the level of drug-induced topoisomerase-mediated lesions and phology by low temperature may result in the partial loss of critical cell death. It has been suggested that strand separation in the presence architecture, microtubule-based vesicular transport, and Golgi func of a stabilized, covalently bound topoisomerase II-drug complex tion in L1210 cells; these losses may contribute toward the protection could lead to erroneous reassociation of topoisomerase II subunits and from ADR acquired at low temperature. The fact that BFA provided a nucleotide tails generating nonhomologous recombination events and consistently higher degree of protection from ADR than nocodazole subsequent alterations in the DNA sequence (20). In accordance, it has may be related to the degree to which each of these agents disrupts been demonstrated that the cytotoxicity of m-AMSA and etoposide various transport processes. For example, microtubules do not appear could be diminished in cells by pretreatment with inhibitors of nucleo to be involved in transporting newly synthesized proteins between the tide synthesis such as aphidicolin and cordycepin (20, 21). However, salvage compartment, Golgi cisternae or fra/w-Golgi (35, 37). Fur the protective effect initiated by inhibitors of nucleotide synthesis has thermore, Golgi units which had been disrupted with nocodazole were not been demonstrated for ADR. Furthermore, the protection that cells shown to retain cis-trans polarity and functionality (42). Therefore, acquired by pretreatment with BFA was not the result of inhibition of although the addition of nocodazole to cells causes the depolymer- nucleotide incorporation into DNA. Treatment of L1210 cells with 10 ization of microtubules, the resulting influence on transport may be /U.MBFAdid not inhibit DNA synthesis. Other laboratories have shown limited to specific interactions such as fusion between early and late that inhibition of protein synthesis (3-5) or depletion of intracellular endosomes (37), disassembly of the Golgi stacks (36), formation of 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 20Â°C,internalization and degradation rates of membrane proteins display a marked decline in activity (27- 29). The exit of proteins from the TON to the cell surface is also significantly reduced below 20Â°C(30, 31). At still lower temperatures, â€¢¿suchas15Â°C,vesicles have been shown to accumulate in an inter mediate compartment between the ER and Golgi (27, 31). Stereologi- cal volume measurements of the Golgi compartment also indicate an increase in the size of the TON at 2()Â°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 high and low temperature, albeit with reduced kinetics at low tem cells/ml and exposed to BFA (10 JIM) at 37Â°Cfor 2 h in 96-well plates (100 Â»Â¿I/well). perature, there may be important regulatory proteins or cofactors [â€¢'HjThymidincwas added at 1 /iCi/well and the cells were incubated for 2 h at 37Â°C. 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 September 27, 2021. © 1993 American Association for Cancer Research. PROTECTION FROM ADRIAMYCIN BY BREFELDIN A the TGN-early endosÃ³me (43), movement of newly synthesized pro shown to protect cells against doxorubicin-induced cytotoxicity (53). teins from the ER to the Golgi or from the Golgi to the plasma It is possible that the disruption of Golgi by BFA results in an eleva membrane, and recycling of receptors (34). As a result, microtubule- tion of GRPs and subsequent protection from ADR similar to that disrupting agents such as nocodazole may not universally inhibit the previously demonstrated (53). Additionally, part of the protective re flow of membrane traffic but have instead been shown to result in the sponse induced by low temperature may be the ability to induce an misdirection of vesicle movement (35). This lack of direction could elevation of GRPs. 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Paul J. Vichi and Thomas R. Tritton

Cancer Res 1993;53:5237-5243.

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