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756 Vol. 11, 756–767, January 15, 2005 Clinical Cancer Research

Intracellular Localization and Trafficking of Fluorescein-Labeled Cisplatin in Human Ovarian Carcinoma Cells

Roohangiz Safaei,1 Kuniyuki Katano,1 INTRODUCTION Barrett J. Larson,1 Goli Samimi,1 Alison K. Holzer,1 The mechanisms that control the cellular accumulation of Wiltrud Naerdemann,1 Mika Tomioka,1,2 cisplatin [cis-diamminedichloroplatinum (DDP)] are poorly understood. Studies of the cellular pharmacology of DDP have Murray Goodman,2 and Stephen B. Howell1 1 2 suggested that specialized membrane-bound proteins mediate the Departments of Medicine, and Chemistry, and the Rebecca and majority of DDP uptake and efflux, and nearly all cell lines John Moores Cancer Center, University of California, San Diego, California selected for resistance to DDP exhibit alterations in drug accumulation (1, 2). Recent studies have identified the copper importer CTR1 (3, 4), and the copper efflux transporters ATP7A ABSTRACT (5), and ATP7B (6, 7), as being important to the cellular Purpose: We sought to identify the subcellular compart- pharmacokinetics and cytotoxicity of DDP. ments in which cisplatin [cis-diamminedichloroplatinum Identification of the conduits along which DDP moves (DDP)] accumulates in human ovarian carcinoma cells and between subcellular compartments after it enters the cell, and the define its export pathways. sites in which it accumulates, has been thus far accomplished Experimental Design: Deconvoluting digital microscopy only indirectly by noting the damage caused by DDP in various was used to identify the subcellular location of fluorescein- organelles or by low-resolution microscopy with energy- labeled DDP (F-DDP) in 2008 ovarian carcinoma cells dispersive X-ray microanalysis (8–10) and electronic probes stained with organelle-specific markers. Drugs that block (11). Electron microscopy (12) can provide the needed vesicle movement were used to map the traffic pattern. resolution for identification of subcellular sites of DDP Results: F-DDP accumulated in vesicles and were not accumulation, but its capability is limited by the relative detectable in the cytoplasm. F-DDP accumulated in the solubility of DDP. Recently, Molenaar et al. (13) showed that Golgi, in vesicles belonging to the secretory export pathway, fluorescein-tagged DDP, abbreviated here as F-DDP, could be and in lysosomes but not in early endosomes. F-DDP used to obtain high-resolution images of the subcellular extensively colocalized with vesicles expressing the copper distribution of DDP. They found that DDP was not uniformly efflux protein, ATP7A, whose expression modulates the distributed in cells but could be detected in many cytoplasmic cellular pharmacology of DDP. Inhibition of vesicle vesicles as well as in the nucleus. trafficking with brefeldin A, wortmannin, or H89 increased In the current study, we used digital deconvoluting the F-DDP content of vesicles associated with the pre-Golgi microscopy in combination with organelle-specific markers to compartments and blocked the loading of F-DDP into examine the intracellular distribution of F-DDP, and employed vesicles of the secretory pathway. The importance of the pathway inhibitory drugs to identify major conduits through secretory pathway was confirmed by showing that which F-DDP traffics as it enters and eventually effluxes from wortmannin and H89 increased whole cell accumulation of cultured human ovarian carcinoma cells. native DDP. Conclusions: F-DDP is extensively sequestered into MATERIALS AND METHODS vesicular structures of the lysosomal, Golgi, and secretory Reagents. Brefeldin A was obtained from ICN Biochem- compartments. Much of the distribution to other compart- icals, Inc. (Aurora, OH), wortmannin from Sigma, Co. (St. ments occurs via vesicle trafficking. F-DDP detection in the Louis, MO), H89 from EMD Biosciences (San Diego, CA), and vesicles of the secretory pathway is consistent with a major media and sera were from Invitrogen (Carlsbad, CA). Antibodies role for this pathway in the efflux of F-DDP and DDP from to TGN38 and ATP7A were purchased from BD Transduction the cell. Laboratories (San Diego, CA), antibodies to early endosomal antigen 1 (EEA1), rab4, and rab11 were from Santa Cruz Received 5/26/04; revised 8/11/04; accepted 9/22/04. Biotechnology, Inc. (Santa Cruz, CA), and the antibody to Grant support: Grants CA78648 and 95298 from the NIH, and grant cMOAT protein 2 (MRP2) was from Alexis Biochemicals (San DAMD17-03-1-0158 from the Department of Defense. This work was Diego, CA). The antibody to golgin97 and specific dyes Alexa conducted in part by the Clayton Foundation for Research, California Flour 647 phalloidin, Hoechst 33342, and LysoTracker Red were Division. Drs. R. Safaei and S.B. Howell are Clayton Foundation obtained from Molecular Probes (Eugene, OR). Texas red– investigators. The costs of publication of this article were defrayed in part by the conjugated secondary antibodies against mouse, rabbit, and goat payment of page charges. This article must therefore be hereby marked antigens were from Jackson ImmunoResearch Laboratories, Inc. advertisement in accordance with 18 U.S.C. Section 1734 solely to (West Grove, PA). F-DDP was synthesized according to a indicate this fact. procedure described elsewhere (14). DDP was a gift from Requests for reprints: Roohangiz Safaei, Department of Medicine 0058, University of California, San Diego, CA 92093. Phone: 858-822- Bristol-Myers Squibb, Co. (Princeton, NJ). 1110; Fax: 858-822-1111; E-mail: [email protected]. Cell Culture and Histochemistry. Ovarian carcinoma #2005 American Association for Cancer Research. 2008 cells (15) and the DDP-resistant subline 2008/C13*5.25

(16) were maintained in RPMI 1640 medium with 10% fetal potassium tetrachloroplatinate to obtain [1-(tBoc-aminomethyl)- bovine serum in humidified air with 5% CO2. Histochemistry 1,2-ehtylenediamine] dichloroplatinum(II). The tBoc group was was done on cells 1 day after seeding them on cover slips. removed with 0.1 N HCl at 70jC and the resulting platinated Labeling with 2 Amol/L F-DDP was done in OptiMEM at 37jC salt was neutralized with 2 N NaOH and then reacted with 5(6)- for 1 hour. Brefeldin A, wortmannin, and H89 were used at 10 carboxyfluorescein preactivated by treatment with ethyldime- Ag/mL, 200 nmol/L, and 2 Amol/L, respectively. All agents were thylaminopropyl carbodiimide and N-hydroxysuccinimide in diluted from freshly made stock solutions in OptiMEM and dimethyl formamide. After centrifugation and successive washes given to cells 15 minutes before and during the 1-hour with water, ethanol, and ether, the final product was character- incubation with 2 Amol/L F-DDP. Lysosomes were labeled in ized for DDP and fluorescein contents by 1H-nuclear magnetic OptiMEM containing 1 Ag/mL of LysoTracker Red for 30 resonance and mass spectrometry. The chemical structure of minutes. All samples were fixed after three quick rinses with F-DDP is presented in Fig. 1 along with the structure of PBS by treatment for 15 minutes with 3.7% formaldehyde in cyclohexylmethylamido fluorescein (CHMA-F), which consists PBS. Samples were permeabilized with 0.3% Triton X-100 in of fluorescein coupled to the linker in the absence of DDP. PBS for 15 minutes, washed three times, 15 minutes each, and F-DDP can become attached to proteins at sites where it is then stained with 1 Ag/mL Hoechst 33324 and 0.4 Ag/mL Alexa sequestered intracellularly by two different mechanisms. In Flour 647 phalloidin, both dissolved in PBS, for 45 minutes and addition to direct reaction of DDP with nucleophilic sites on then mounted on glass slides, or processed for immunostaining. intracellular molecules, in the presence of formaldehyde, F-DDP Immunostaining was done after a 1-hour incubation of fixed and can also become cross-linked to immediately adjacent proteins permeabilized samples with 0.3% bovine serum albumin. via the CHMA-F moiety. To show that F-DDP does in fact Primary antibodies were diluted in PBS containing 0.3% bovine become firmly bound by one or the other of these mechanisms to serum albumin. Cells were then washed three times, 15 minutes proteins or other molecules at sites where it is concentrated, 2008 each, with PBS and incubated for 1 hour with secondary cells were incubated with 2 Amol/L F-DDP for 1 hour, fixed with antibodies in PBS and 0.3% bovine serum albumin, followed by 3.7% formaldehyde in PBS and treated with increasing three 3, 15-minute washes in PBS and mounting on glass slides. concentrations of Triton X-100 for 15 minutes prior to being Mounting medium contained 24 g polyvinyl alcohol, 60 g stained with antibodies to specific structural markers. As shown glycerol, 60 mL H2O, 120 mL 0.2 mol/L Tris-HCl (pH 8.5). in Fig. 2, F-DDP remained associated with the subcellular Microscopy was done at the University of California, San structures into which it was initially sequestered even after Diego Cancer Center Digital Imaging Shared Resource using a treatment with 1% Triton X-100, demonstrating that F-DDP DeltaVision deconvoluting microscope system (Applied Preci- binds cellular components very firmly under these fixation sion, Inc., Issaquah, WA). Images were captured from 0.2 A conditions. Examination of a large number of cells indicated that sections by 100Â and 40Â lenses and SoftWorx software the differences in vesicle size and distribution apparent in Fig. 2 (Applied Precision) was used for deconvoluting data. Image reflected cell-to-cell heterogeneity rather than an effect of quantification was done with Data Inspector program in increasing Triton X concentration. The heterogeneity in staining SoftWorx or by NearCount software. pattern is likely to be related to differences in the flux of Measurement of Platinum Accumulation. One million vesicular trafficking which may be associated with such factors cells were seeded in 35 mm dishes and grown until they were as cell cycle phase. 75% to 80% confluent. The medium was replaced by 1 mL of To determine the extent to which F-DDP mimics the fresh medium containing 2 Amol/L DDP or DDP plus inhibitory behavior of DDP, F-DDP, CHMA-F, and DDP were compared drugs, and the cells were incubated at 37jC. At the requisite time with regard to their cytotoxicity, cross-resistance, and accumu- points, the medium was poured off and the dishes were quickly lation levels using the 2008 DDP-sensitive parental human rinsed thrice with ice-cold PBS and then 215 AL 70% nitric acid ovarian carcinoma cell line and the 2008/C13*5.25 DDP- was added to each plate. The cell lysates were then transferred resistant subline. Both DDP and F-DDP showed concentration- into 15 mL centrifuge tubes, capped and placed at 65jCfor2 dependent cytotoxicity that was greater for 2008 than for the hours, after which the samples were diluted with an indium (Acros Organics, Tustin, CA) solution to a final concentration of 5% acid and 1 ppb of indium. Platinum was measured with a Thermo Finnigan inductively coupled plasma mass spectrometer (model element 2) at the Analytical Facility at the Scripps Institute of Oceanography. Platinum values were normalized to the protein content measured by the Bradford method using the Bio-Rad kit (Hercules, CA).

RESULTS Synthesis and Characterization of F-DDP and CHMA-F. F-DDP, consisting of DDP to which a fluorescein was linked, was synthesized using a modification of the method described by Molenaar et al. (13). Briefly N3-tBoc-1,2,3-propanetriamine was prepared from 3-amino-1,2-propanediol and then reacted with Fig. 1 Structure of F-DDP and CHMA-F.

Fig. 2 Effect of permeabilization with different concentrations of Triton X-100 on F-DDP association with subcellular structures in 2008 cells. F-DDP-labeled cells were fixed as described and then incubated with 0.0%, 0.3%, 0.6%, and 1% Triton X-100 in PBS for 15 minutes prior to being stained with Alexa Flour 647 phalloidin (red) to identify filamentous actin, and Hoechst 33342 (blue) to stain nuclei. F-DDP (green).

2008/C13*5.25 cells, whereas CHMA-F was not toxic even at a structures and less intense fluorescence in the nucleus (Fig. 3E concentration of 500 Amol/L. The ratio of the IC50 values for the and F). Although there was substantial variability in the 2008/C13*5.25 and 2008 cells, determined by clonogenic assay, intensity of F-DDP fluorescence between cells of a given field, was similar for both compounds, being 5.3 Amole/L for DDP and a 1-hour exposure was sufficient to produce detectable 5.7 Amole/L for F-DDP. As previously documented for DDP fluorescence in the majority of cells in single preparation. (14), the accumulation of F-DDP was reduced in the 2008/ Consistent with the observations made by Molenaar et al. (13), C13*5.25 cells to 26% of that in the 2008 cells at 1 hour. Thus, F-DDP in the nucleus exhibited a finely granular pattern, F-DDP is subject to the same mechanisms that render cells whereas elsewhere in the cell, F-DDP was distributed in discrete resistant to DDP. vesicular structures rather than being diffusely localized Additional evidence that F-DDP mimics the behavior of throughout the cytoplasm (Fig. 3E). A serial analysis of the DDP was provided by analyses of the subcellular distribution change in intracellular distribution of F-DDP over the first 30 of F-DDP and CHMA-F in 2008 cells. Figure 3 shows that minutes of exposure is presented in Fig. 4. When F-DDP was although CHMA-F was distributed in structures that extensively applied to the cells and then immediately washed away (time 0) colocalized with filamentous actin in 2008 cells (A), the there was intense fluorescence at the cell surface but little distribution of F-DDP was quite different (C and E). F-DDP activity elsewhere in the cell. By 2 to 5 minutes, fluorescence was localized to discrete perinuclear vesicular structures and the appeared in cytoplasmic organelles, and then over the ensuing nucleus. This indicates that the subcellular distribution of F-DDP 10 to 30 minutes it appeared in nuclear structures. is driven predominantly by the DDP moiety rather than the Colocalization of F-DDP with Endosomal/Lysosomal CHMA-F fluorophore in the F-DDP complex. Furthermore, as Markers. Antibodies to the EEA1, rab4, and rab11 were used shown in Fig. 3B, D, and F, the distribution of CHMA-F was to identify the early (17), fast, and slow recycling (18) endo- also markedly different from that of F-DDP in the DDP-resistant somes, respectively. LysoTracker Red was used to identify the 2008/C13*5.25 cells. It is also clear that the compartmental acidic lysosomal structures. Figure 5A shows representative distribution of F-DDP in the DDP-sensitive 2008 and DDP- images from studies in which 2008 cells were exposed to resistant 2008/C13*5.25 is quite different. The basis for this is F-DDP and then stained with anti-EEA1. There was very not currently known but is under active investigation. little colocalization of F-DDP with EEA1 at any point up to Subcellular Distribution of F-DDP. As shown in Fig. 3, 60 minutes of exposure to F-DDP. Likewise, as shown in incubation of cells with 2 Amol/L F-DDP for 1 hour produced Fig. 5B, there was no detectable colocalization of F-DDP with readily detectable fluorescence in a variety of cytoplasmic rab4. Staining of F-DDP-labeled cells with antibodies against

Fig. 3 Distribution of F-DDP and CHMA-F in 2008 ovarian carcinoma cells. Cells were incubated with 2 Amol/L of either F-DDP or CHMA-F for 1 hour in parallel, and then stained for filamentous actin (red) with Alexa Flour 647 phalloidin and with Hoechst 33342 for nuclei (blue) prior to being imaged for F-DDP (green) accumulation. A, distribution of CMHA-F in DDP-sensitive 2008 cells; B, distribution of CMHA-F in DDP-resistant 2008/C13*5.25 cells; C, distribution of F-DDP in DDP-sensitive 2008 cells; D, distribution of F-DDP in DDP-resistant 2008/ C13*5.25 cells; E, distribution of F-DDP in a single 2008 cell; F, distribution of F-DDP in a single 2008/C13*5.25 cell.

the small GTPase rab11, which is a marker for the slowly re- that these vesicles may be part of the lysosomal vesicular cycling endosomal compartment (18), showed limited and weak secretory pathway. colocalization between the two fluorescent signals as shown in Association of F-DDP with the Golgi and Trans - Golgi Fig. 5C. Thus, there seems to be little transit of F-DDP through Network. To examine the involvement of the Golgi apparatus the early endosomal compartment during accumulation in the in F-DDP trafficking, 2008 cells were exposed to 2 Mmol/L cell. F-DDP did not enter endosomes that recycle rapidly back F-DDP for 1 hour and then stained with a monoclonal antibody from the early endosome to the cell surface and entered slowly against the grip domain Golgi protein, golgin97 (19). Golgin97 recycling endosomes only to a minimal degree. localizes to the trans side of the Golgi apparatus and is In contrast to the early and recycling endosomal believed to participate in the trafficking of vesicles between the compartments, F-DDP accumulated extensively in lysosomal Golgi stacks and the trans-Golgi network (20). As shown in vesicles identified on the basis of their accumulation of Fig. 6A, a subset of the golgin97-positive vesicles accumulated LysoTracker Red as shown in Fig. 5D. Strong colocalization F-DDP and these were scattered throughout the cytoplasm. between F-DDP and LysoTracker Red was especially In contrast, as shown in Fig. 6B, there was no detectable pronounced in regions near the plasma membrane, suggesting colocalization of F-DDP with the trans-Golgi network marker

TGN38, a protein found predominantly in the tubulovesicular Colocalization with ATP7A and MRP2. Antibodies subcompartment of the trans-Golgi network (21). These results against two markers of vesicles belonging to the secretory show the involvement of golgin97-positive Golgi-derived pathway, MRP2 and ATP7A, both of which have been im- vesicles in the trafficking of F-DDP, and indicate that F-DDP plicated in the transport of DDP (22, 23), showed extensive is directed in a selective manner to downstream compartments colocalization with F-DDP. As shown in Fig. 6B, vesicles other than the TGN38-positive subcompartment of the trans- exhibiting colocalization of F-DDP with ATP7A were found Golgi network. It is of interest that F-DDP colocalized with predominantly in a perinuclear distribution, although some golgin97-positive vesicles found not just in the region of the vesicles were dispersed throughout the cell. As shown in Golgi and trans-Golgi network but also out at the periphery of Fig. 6C, vesicles exhibiting colocalization of F-DDP and MRP2 the cell. This suggests that either F-DDP is sequestered into the were located mainly in a perinuclear distribution. NearCount Golgi first and then carried over long distances, or that F-DDP software determined that 64 F 2% (mean F SE) of total enters these vesicles after they have departed the Golgi and cytoplasmic F-DDP colocalized with ATP7A and 64 F 2% moved toward the periphery of the cell. (mean F SE) with MRP2 in 2008 cells. The extensive

Fig. 4 Variation in the distribution of F-DDP as a function of time in 2008 cells. Cells were exposed to 2 Amol/L F-DDP for 0, 2, 5, 10, and 30 minutes, fixed and costained with Alexa Flour 647 phalloidin to localize the actin filaments (artificially colored red). Yellow, overlapping of the F-DDP signal with that for actin.

Fig. 5 Colocalization of F-DDP (green) with specific compartmental markers (red) in 2008 cells incubated with 2 Amol/L F-DDP for 1 hour. A, colocalization with EEA1-positive early endosomal vesicles; B, colocalization of F-DDP with rab4-positive recycling endosomes; C, colocalization of F-DDP with rab11-positive recycling endosomes; D, colocalization of F-DDP with lysosomal marker LysoTracker Red. Yellow, structure is positive for both F-DDP and organelle markers. Nuclei are stained with Hoechst 33342 (blue).

association of F-DDP with vesicles of the export pathway sug- cause intermixing of Golgi components with those of the ER as gests that ATP7A and MRP2, or closely associated proteins, are well as fusion of endosomes, lysosomes, and the trans-Golgi involved in the efflux of this drug. network (27). As expected, treatment of 2008 cells with Effect of Pathway Inhibitory Drugs on F-DDP Localiza- brefeldin A caused Golgi fragmentation and collapse of tion. To further define the role of each vesicular compartment golgin97-positive structures toward the perinuclear region in the trafficking of F-DDP, 2008 cells were treated with F-DDP (Fig. 7A). However, the golgin97-positive vesicles in brefeldin and agents that selectively block various trafficking pathways. A–treated cells were almost completely devoid of F-DDP, Brefeldin A was used to inhibit initial steps of the vesicular indicating that the blockade of ER to Golgi movement also secretory pathway and wortmannin was used to inhibit PI3 blocked the loading of F-DDP into these vesicles, presumably kinase-mediated fusion of vesicles along the pathway from due to incorrect localization of those proteins normally endosomes to lysosomes (24). The protein kinase A inhibitor responsible for sequestering F-DDP. This conclusion was H89 was used to block post-Golgi secretory events (25). confirmed by the results of an experiment in which brefeldin Blockade of vesicle movement from the endoplasmic A and F-DDP-treated cells were stained with antibodies against reticulum to Golgi stacks with brefeldin A (26) is known to MRP2 and ATP7A. As shown in Fig. 7B and C, the ATP7A- and

Fig. 6 Colocalization of F-DDP (green) with vesicles expressing specific markers (red) for (A) golgin97, (B) ATP7A, and (C) MRP2. Yellow, structure is positive for both F-DDP and organelle markers. Nuclei are stained with Hoechst 33342 (blue).

Fig. 7 Effect of brefeldin A, wortmannin, and H89 on the subcellular distribution of F-DDP. A, effect on colocalization of F-DDP (green) with golgin97 (red). B, effect on colocalization of F-DDP with vesicles expressing ATP7A (red).

Fig. 7 (cont’d) C, effect on colocalization of F-DDP with vesicles expressing MRP2 (red). D, effect on colocalization of F-DDP with vesicles expressing TGN38 (red). Yellow, the structure is positive for both the F-DDP and organelle markers.

MRP2-expressing vesicles were also completely devoid of wortmannin and H89 on whole cell platinum accumulation was F-DDP in brefeldin A–treated cells. Thus, failure to load determined by inductively coupled plasma mass spectroscopy. F-DDP into upstream vesicles associated with the trans-Golgi At a concentration of 200 nmol/L wortmannin, increased whole network was accompanied by a lack of F-DDP in the more cell uptake by 6.0 F 0.2-fold (mean F SE; P < 0.001). H89 downstream vesicles of the vesicle secretory pathway. Interest- produced a similar effect and increased whole cell DDP uptake ingly, treatment of cells with brefeldin A resulted in some degree by 7.5 F 0.1-fold (mean F SE; P < 0.001). Thus, wortmannin of colocalization between F-DDP and the TGN38 marker and H89 produced similar effects on the accumulation of F- (Fig. 7D) presumably due to the dissolution of TGN38- DDP and native DDP. containing vesicles into the endosomal/lysosomal compartment (27). This indicated that the Golgi and trans-Golgi compartments are downstream of the endosomal compartments in the DISCUSSION intracellular transport of F-DDP. In this study a fluorescent form of DDP was used to identify At 200 nmol/L wortmannin is reported to inhibit the late the subcellular compartments into which the drug is sequestered stages of the endosomal fusion and the maturation of endosomes and through which it traffics. The results confirm and extend our into lysosomes (24). At this concentration, wortmannin is previous work showing that F-DDP mimics DDP with respect relatively specific for inhibition of the PI3 kinases involved in to cytotoxicity, accumulation pattern, and colocalization with the vesicular trafficking (28). Treatment of 2008 cells with DDP export protein ATP7B (14). The finding that the majority of wortmannin increased the overall uptake of F-DDP: quantifica- the intracellular F-DDP was associated with vesicular structures tion of raw digital images with Softworks software indicated confirms the observation of Molenaar et al. (13) that, instead of that the mean F-DDP fluorescence per cell was increased by diffusing freely through the cytoplasm, F-DDP is sequestered 27 F 1% (SE; P = 5.62 Â 10À28). However, wortmannin into specific families of vesicles presumably by specialized prevented the sequestration of F-DDP into golgin97-expressing membrane-bound proteins that mediate the transport of DDP. vesicles of the Golgi or vesicles of the secretory pathway The results presented here provide a small-scale map of the expressing MRP2 or ATP7A (Fig. 7A-C). In the presence of vesicular pathways involved in the movement of the drug wortmannin, the structures that contained golgin97 were not through the human ovarian carcinoma cells, and information only devoid of F-DDP but also localized to a small corner near about which compartments are involved in the early and later the nucleus (Fig. 7A). Interestingly, as wortmannin blocked steps of this process. the flux of F-DDP into the vesicles of the secretory pathway, Several lines of evidence indicate that labeling of DDP with it caused F-DDP to backup into the more upstream TGN38- fluorescein does not change those components of the cellular positive vesicles that in the absence of wortmannin did not pharmacology of the drug that are central to the DDP-resistant accumulate F-DDP at all (Fig. 7D). These data indicate that the phenotype. First, the resistant cells accumulate less F-DDP in endosomal/lysosomal system is involved in directing F-DDP into parallel to their failure to accumulate DDP. Second, despite the the vesicle of the secretory pathway. fact that F-DDP is less potent than DDP, it retains substantial The isoquinolinesulfonamide protein kinase inhibitor H89 is cytotoxicity and the DDP-resistant cells are as resistant to known to block steps downstream of the Golgi vesicles within the F-DDP as they are to DDP. Thus, mechanisms essential to the secretory pathway (29). By blocking COPII recruitment and DDP-resistant phenotype are operative on F-DDP as well as export from the endoplasmic reticulum, H89 causes the collapse DDP. Third, the subcellular distribution of F-DDP is markedly of the endoplasmic reticulum–associated Golgi compartment different from that of the fluorophore, CHMA-F, indicating that (30, 31) allowing proteins resident in the Golgi to remain the presence of the DDP component in F-DDP predominates juxtanuclear (31, 32). Treatment of 2008 cells with H89 produced over the effect of the CHMA-F moiety. Finally, F-DDP showed the expected juxtanuclear localization of the golgin97 protein and localization in intracellular structures known to accumulate DDP increased total cellular F-DDP fluorescence (Fig. 7A), but that the on the basis of direct measurements of platinum content, same time blocked the accumulation of F-DDP in vesicles including the mitochondria (33). expressing ATP7A or MRP2 (Fig. 7B and C). As was observed F-DDP initially becomes sequestered into the lysosomes with wortmannin, H89 caused the backing-up of F-DDP into and is subsequently found in vesicles derived from the Golgi TGN38-positive vesicles that were found in a well-defined ring that participate in the vesicle of the secretory pathway. around the nucleus (Fig. 7D). These results are consistent with a Endosomes that express EEA1 protein constitute the first role for a protein kinase in the loading of F-DDP into vesicles intracellular station for endocytic vesicles derived from the destined for subsequent export from 2008 cells. plasma membrane (34). The near absence of F-DDP from Effect of Pathway Inhibitory Drugs on Whole Cell these early endosomes indicates that the uptake of F-DDP is Accumulation of DDP. The studies with digital deconvoluting not mediated by the endocytic pathways that deliver their microscopy showed that wortmannin and H89 increased whole cargo to this station. Similarly, vesicles expressing rab4 do not cell accumulation of F-DDP and caused excessive accumula- seem to function in the flux of F-DDP in 2008 cells. tion in vesicles at the upper end of the pathway, at the same However, a role for recycling vesicles expressing rab11 cannot time reducing the amount of F-DDP founding vesicles at the be completely ruled out because there was a small degree of lower end of the vesicle secretory pathway. To confirm that colocalization between F-DDP and antibodies to rab11. The these drugs produced the same effect on the cellular rab11 GTPase has been localized to recycling endosomes and pharmacology of native DDP, 2008 cells were treated for 1 the trans-Golgi network and is required for transport of hour with 2 Amol/L DDP instead of F-DDP and the effect of recycling endosomes back to the plasma membrane (35–37).

It is possible that some of the F-DDP that reaches the vesicle of the secretory pathway is delivered by rab11-positive vesicles. Accumulation of F-DDP in lysosomes is consistent with the reported role of this compartment in the regulation of tumor cell sensitivity to DDP (38, 39). Lysosomes are at the junction of both biosynthetic and the degradative pathways. They receive their enzymes and polysaccharide membrane proteins from the trans-Golgi network mostly via multivesicular bodies which also sort the endocytosed proteins for recycling or degradation by lysosomes (reviewed by Katzmann et al. 40). It is possible that some of the F-DDP found in this compartment became associated with proteins elsewhere, and which were then directed to lysosomes for degradation. Whether any of the F-DDP or DDP that becomes sequestered Fig. 8 Schematic diagrams showing the extent of F-DDP accumulation in lysosomes finds its way to the cytoplasm remains to be in various subcellular compartments and trafficking from upstream determined. However, lysosomes do possess metal transporters compartments to the vesicle secretory pathway in 2008 cells. Black, vesicles with the highest accumulation of F-DDP; gray, lower-level such as NRAMP2, which functions to export iron from F-DDP accumulation; white, no accumulation. lysosomes (41). It is thus likely that transporters capable of exporting DDP from lysosomes would also be present in this compartment. 23, 38, 45). F-DDP becomes associated with lysosomes and One step in the trafficking of F-DDP seems to involve the golgin97-expressing vesicles of the Golgi and is also found in trans side of the Golgi, marked by the grip domain protein vesicles that express markers identifying them as belonging to golgin97. This protein is proposed to function in tethering of the secretory pathway that eventually exports vesicles from the docking of vesicles (19). Golgi-derived vesicles containing cell. Disruption of the secretory pathway with brefeldin A, F-DDP seem to traffic towards the cell surface, at the same time wortmannin, or H89 produced effects consistent with the retaining the golgin97 protein. The Golgi is also reported to be hypothesis that F-DDP first enters an upstream compartment the site of origin for vesicles destined for export from the cell via and is then transferred to the vesicle secretory pathway and the secretory pathway that express MDR1 (42) and MRP2 (42). hence out of the cell. Whether this upstream compartment Thus, the finding of F-DDP in both golgin97 and MRP2-positive consists solely of components of the Golgi, the lysosomes, vesicles is consistent with the proposal F-DDP passes through or both cannot be determined with the available data, although the Golgi and is then directed to vesicles belonging to the the fact that wortmannin prevented loading of F-DDP into secretory pathway. Further evidence is provided by the golgin97-positive vesicles suggests that the flux of F-DDP is observation that F-DDP becomes associated with vesicles predominantly from the lysosomal compartment toward the expressing ATP7A, another marker of vesicles belonging to Golgi. These drugs increased total cellular F-DDP, increased the secretory pathway (43). the F-DDP content of vesicles associated with the lysosomes The association of F-DDP with MRP2 is consistent with a at the upstream end of the pathway, and decreased the flux of previous study suggesting that MRP2 plays a role in the efflux F-DDP into vesicles at the downstream end of the secretory of DDP (44). It is noteworthy that the majority of MRP2- pathway. The accumulation of F-DDP in vesicles expressing expressing vesicles that contained F-DDP were located deep in TGN38 in the brefeldin A and wortmannin-treated cells the interior of the cells and not at the plasma membrane. This suggests that inhibition of the delivery of F-DDP from the suggests that, in these carcinoma cells that lack polarity, MRP2 prelysosomal pathway to downstream compartments caused functions to sequester F-DDP, or its glutathione conjugates, the backup of F-DDP into an upstream compartment which into intracellular vesicles that are subsequently exported rather also receives input from the trans-Golgi network. The concept than pumping F-DDP directly out of the cell across the plasma that a substantial fraction of the efflux of DDP is mediated membrane. by sequestration into vesicles that are then exported from The substantial degree of colocalization between F-DDP the cell is further supported by the fact that wortmannin and and the copper efflux transporter ATP7A is of particular interest H89 increased whole cell accumulation of both F-DDP and given that ATP7A has now been shown to control the cellular native DDP. pharmacology of DDP and sensitivity to the cytotoxic effect of The finding of high levels of F-DDP in lysosomes is of this drug (23, 45). Many malignant cells also express the other particular interest given the observation that cells selected for copper efflux transporter ATP7B, and ATP7B has also been DDP resistance have major defects in lysosomal function shown to modulate the cellular pharmacology of DDP and (38, 46). The fact that F-DDP did not colocalize with EEA1- sensitivity to its cytotoxic effect (2, 6). Recently ATP7B has expressing precursor early endosomes suggests that F-DDP been shown to colocalize with F-DDP in vesicles of the secretory enters the lysosomes via another uptake pathway, perhaps via a pathway (14). pinocytotic mechanism (47). Further work is required to Figure 8 presents a schematic diagram of the trafficking determine the exact sequence of drug sequestration into the of F-DDP in human ovarian carcinoma cells and summarizes lysosomes and the mode of its delivery to the secretory the available information from this and prior studies (6, 13, 14, compartment.

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