Intracellular Localization and Trafficking of Fluorescein-Labeled Cisplatin in Human Ovarian Carcinoma Cells Roohangiz Safaei, 1 Kuniyuki Katano, 1 Barrett J. Larson, 1 Goli Samimi, 1 Alison K. Holzer, 1 Wiltrud Naerdemann, 1 Mika Tomioka, 1,2 Murray Goodman, 2 and Stephen B. Howell 1 Departments of 1 Medicine, and 2 Chemistry, and the Rebecca and John Moores Cancer Center, University of California, San Diego, California ABSTRACT Purpose: We sought to identify the subcellular compart- ments in which cisplatin [cis -diamminedichloroplatinum (DDP)] accumulates in human ovarian carcinoma cells and define its export pathways. Experimental Design: Deconvoluting digital microscopy was used to identify the subcellular location of fluorescein- labeled DDP (F-DDP) in 2008 ovarian carcinoma cells stained with organelle-specific markers. Drugs that block vesicle movement were used to map the traffic pattern. Results: F-DDP accumulated in vesicles and were not detectable in the cytoplasm. F-DDP accumulated in the Golgi, in vesicles belonging to the secretory export pathway, and in lysosomes but not in early endosomes. F-DDP extensively colocalized with vesicles expressing the copper efflux protein, ATP7A, whose expression modulates the cellular pharmacology of DDP. Inhibition of vesicle trafficking with brefeldin A, wortmannin, or H89 increased the F-DDP content of vesicles associated with the pre-Golgi compartments and blocked the loading of F-DDP into vesicles of the secretory pathway. The importance of the secretory pathway was confirmed by showing that wortmannin and H89 increased whole cell accumulation of native DDP. Conclusions: F-DDP is extensively sequestered into vesicular structures of the lysosomal, Golgi, and secretory compartments. Much of the distribution to other compart- ments occurs via vesicle trafficking. F-DDP detection in the vesicles of the secretory pathway is consistent with a major role for this pathway in the efflux of F-DDP and DDP from the cell. INTRODUCTION The mechanisms that control the cellular accumulation of cisplatin [cis -diamminedichloroplatinum (DDP)] are poorly understood. Studies of the cellular pharmacology of DDP have suggested that specialized membrane-bound proteins mediate the majority of DDP uptake and efflux, and nearly all cell lines 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 (5), and ATP7B (6, 7), as being important to the cellular pharmacokinetics and cytotoxicity of DDP. Identification of the conduits along which DDP moves between subcellular compartments after it enters the cell, and the sites in which it accumulates, has been thus far accomplished only indirectly by noting the damage caused by DDP in various organelles or by low-resolution microscopy with energy- dispersive X-ray microanalysis (8 – 10) and electronic probes (11). Electron microscopy (12) can provide the needed resolution for identification of subcellular sites of DDP accumulation, but its capability is limited by the relative solubility of DDP. Recently, Molenaar et al. (13) showed that fluorescein-tagged DDP, abbreviated here as F-DDP, could be used to obtain high-resolution images of the subcellular distribution of DDP. They found that DDP was not uniformly distributed in cells but could be detected in many cytoplasmic vesicles as well as in the nucleus. In the current study, we used digital deconvoluting microscopy in combination with organelle-specific markers to examine the intracellular distribution of F-DDP, and employed pathway inhibitory drugs to identify major conduits through which F-DDP traffics as it enters and eventually effluxes from cultured human ovarian carcinoma cells. MATERIALS AND METHODS Reagents. Brefeldin A was obtained from ICN Biochem- icals, Inc. (Aurora, OH), wortmannin from Sigma, Co. (St. Louis, MO), H89 from EMD Biosciences (San Diego, CA), and media and sera were from Invitrogen (Carlsbad, CA). Antibodies to TGN38 and ATP7A were purchased from BD Transduction Laboratories (San Diego, CA), antibodies to early endosomal antigen 1 (EEA1), rab4, and rab11 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and the antibody to cMOAT protein 2 (MRP2) was from Alexis Biochemicals (San Diego, CA). The antibody to golgin97 and specific dyes Alexa Flour 647 phalloidin, Hoechst 33342, and LysoTracker Red were obtained from Molecular Probes (Eugene, OR). Texas red– conjugated secondary antibodies against mouse, rabbit, and goat antigens were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). F-DDP was synthesized according to a procedure described elsewhere (14). DDP was a gift from Bristol-Myers Squibb, Co. (Princeton, NJ). Cell Culture and Histochemistry. Ovarian carcinoma 2008 cells (15) and the DDP-resistant subline 2008/C13*5.25 Received 5/26/04; revised 8/11/04; accepted 9/22/04. Grant support: Grants CA78648 and 95298 from the NIH, and grant DAMD17-03-1-0158 from the Department of Defense. This work was conducted in part by the Clayton Foundation for Research, California Division. Drs. R. Safaei and S.B. Howell are Clayton Foundation investigators. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Roohangiz Safaei, Department of Medicine 0058, University of California, San Diego, CA 92093. Phone: 858-822- 1110; Fax: 858-822-1111; E-mail: rsafaei@ucsd.edu. # 2005 American Association for Cancer Research. Vol. 11, 756–767, January 15, 2005 Clinical Cancer Research 756 Cancer Research. on December 14, 2021. © 2005 American Association for clincancerres.aacrjournals.org Downloaded from