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Increased Rhodamine 123 Uptake by Carcinoma Cells1

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Increased Rhodamine 123 Uptake by Carcinoma Cells1 [CANCER RESEARCH 45, 6093-6099, December 1985] Increased Rhodamine 123 Uptake by Carcinoma Cells1 KarenK.Nadakavukaren,2JohnJ.Nadakavukaren,andLanBoChen3 Dana Farber Cancer Institute, Boston, Massachusetts 02115, and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115 ABSTRACT and retention of this dye by mitochondria apparently correlates with the mitochondrial membrane potential (4). Rh-123 has been The total cellular content of the fluorescent mitochondrial- utilized as an indicator of mitochondrial membrane potential in specific dye rhodamine 123 (Rh-123) was quantified by butanol studies of cells undergoing alterations in metabolic state (5-8). extraction as a function of time of exposure and dose for a Rh-123 may also demonstrate a qualitative difference between variety of cell lines. These results were compared with observa the mitochondria of normal and transformed cells. Johnson ef al. tions made by fluorescence microscopy on dye localization and (9) observed that mitochondria of feline sarcoma virus-trans mitochondrial morphology. There appeared to be two categories formed mink fibroblasts retain significantly less Rh-123 after of cell types based on Rh-123 uptake: those which progressively transfer to dye-free medium than do mitochondria of the untrans- accumulate the dye, such as Ehrlich ascites tumor cells, carci formed parent cells. A more dramatic difference was reported by noma-derived lines MCF-7, PaCa-2, EJ, HeLa, and normal fibro- Summerhayes ef a/. (10), who found that mitochondria of most blast line CCL 64; and those which appear to equilibrate with carcinoma-derived cells retain Rh-123 for much longer periods the extracellular dye within 1 h of incubation in Rh-123 (1 M9/ml) than do mitochondria of normal epithelial-derived cells. This with a minimal level of uptake, such as the normal epithelial- phenomenon appears to correlate with selective killing of carci derived lines CV-1 and MDCK and the transformed fibroblast line noma cells in vitro, as well as in vivo (11-13). The mechanism 64F3. Within the first category, the absolute value of uptake per responsible for the difference in interaction of Rh-123 with car cell correlated with the concentration of Rh-123 in the medium cinoma and normal epithelial-derived cells is not understood. and with the period of exposure to the dye up to a point of We undertook to examine the first step in this interaction, apparent cellular saturation. The length of time required for namely, the uptake of Rh-123 into the cell. Although cellular Rh- apparent saturation depended on the cell type. In the second 123 content can be monitored in terms of relative fluorescence category equilibration was very early, and the total uptake was by flow cytometry and microfluorimetry, it is difficult to make a function of the extracellular concentration of Rh-123. This quantitative measurements using these techniques. We were probably does not represent a saturation level of dye content in especially interested in quantifying the uptake of Rh-123 in the non-accumulating, low uptake cell lines. Fluorescence mi cultures of attached cells as a function of dose and exposure croscopy revealed that Rh-123 localization was initially mito- time. Here we report a reproducible procedure for measuring chondrial-specific for all of the cell lines examined. Over time, total cellular Rh-123 uptake using the butanol extraction method. alterations in mitochondrial morphology and cytoplasmic fluores These experiments included observations by fluorescence mi cence were observed in the high uptake cell lines but not in the croscopy to monitor intracellular Rh-123 localization. The results minimal uptake cell lines. Incubation of the high uptake HeLa cell suggest that Rh-123 uptake among various cell lines can differ line with the mitochondrial membrane potential inhibitor p- both quantitatively and qualitatively and may in fact indicate trifluoromethoxyphenylhydrazone substantially decreased Rh- differing mechanisms of uptake. 123 uptake. These observations may indicate a transformation- related characteristic of carcinoma cell mitochondria. It may be MATERIALS AND METHODS possible to exploit the mechanism responsible for the progres sive accumulation of Rh-123 by carcinoma-derived cell types for Cells. Cell lines were obtained as follows: normal mink lung cell line chemotherapeutic approaches to certain types of carcinomas. (CCL64) and feline sarcoma virus transformant (64F3) were from M. Essex (Harvard School of Public Health); normal canine kidney epithelial line (MDCK), Ehrlich-Lettre mouse Ascites carcinoma line (CCL77), and INTRODUCTION human pancreatic carcinoma line (PaCa-2) were from the American Type Culture Collection (Rockville, MD); human bladder carcinoma (EJ) was The permeant cationic fluorochrome Rh-123" has been used provided by Dr. Ian Summerhayes (Dana Farber Cancer Institute, Boston, in a variety of studies of mitochondria in living cells (reviewed in MA); and human breast carcinoma line (MCF-7) was from Dr. M. Rich Refs. 1 and 2). At non-toxic concentrations, Rh-123 provides (Michigan Cancer Foundation). Cells were grown in DME supplemented low background, high resolution fluorescent images of mitochon with either 5 or 10% calf serum (Grand Island Biological Co., Gaithers- dria in a variety of cell types (3). The preferential accumulation burg, MD). Chemicals. Rh-123 was obtained from Eastman Organic Chemicals Received 3/28/85; revised 8/14/85; accepted 8/19/85. (Rochester, NY). A 10 mg/ml stock solution in dimethyl sulfoxide was 1Supported by grants from the National Cancer Institute and the Council for kept in the dark at 4°Cand used for the preparation of all solutions and Tobacco Research. 'Partially supported by a USPHS training grant awarded to A. B. Pardee. standards used in this study. FCCP was obtained from Sigma Chemical Company (St. Louis, MO) and was stored at 4°Cas a stock in dimethyl Present address: Institute of Molecular Biology, University of Oregon, Eugene, OR 97403. sulfoxide. Butanol (fluorometric analysis grade 1-butanol) was obtained 3 Recipient of an American Cancer Society Faculty Research Award. To whom from Fisher (Medford, MA). requests for reprints should be addressed, at Dana Farber Cancer Institute, 44 Monitoring of Rhodamine 123 Uptake by Fluorescence Micros Binney St., Boston, MA 02115. 4 The abbreviations used are: Rh-123, rhodamine 123; DME, Dulbecco's modi copy. Subconfluent cultures of cells on 12-mm round glass coverslips fied Eagle's medium; FCCP, p-trifluoromethoxyphenylhydrazone. (Rochester Scientific, Rochester, NY) were incubated at 37°C in DME CANCER RESEARCH VOL. 45 DECEMBER 1985 6093 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1985 American Association for Cancer Research. CELLULAR UPTAKE OF RHODAMINE 123 with 5% calf serum containing the desired concentration of Rh-123 sity can be affected by metal ions or dye concentration and by (usually 1 ^g/rnl). A coverslip was removed and the cells were observed the polarity of the environment. pH levels may alter the chemical at hourly intervals. Coverslips were washed by dipping in three succes form of the dye (ionization, dimerization, etc.), and salts can sive baths of fresh medium without Rh-123 for approximately 5 s per cause spectral shifts. A red shift in the fluorescence emission of bath and were mounted on a drop of fresh medium on a live cell Rh-123 within the mitochondria of living cells has been reported observation chamber as described (3). Epifluorescent microscopy and photography were performed essentially as described (3), except that a (6). Rather than measure Rh-123 fluorescence within the cellular neutral density filter was used routinely in the excitation beam to reduce environment, butanol extraction was used to effect the rapid and bleaching and other photo-induced effects during observation. A photo efficient solubilization of Rh-123 from all cellular compartments. graphic record of relative differences in fluorescent intensity was made With this method, more than 95% of the dye is extracted from by manual exposure for 2 and 4 s of several representative fields without the culture dish. Prolonging the incubation time with butanol by a neutral density filter, at hourly time points. Monitoring of Rh-123 Uptake by Butano! Extraction. Cellular Rh- 50% did not significantly increase the amount of dye extracted (data not shown). Solubilizing Rh-123 in butanol, which is a 123 uptake is defined here as the amount of dye, calculated on a per cell basis, which is solubilized from cell cultures by butanol immediately uniform, nonpolar solvent, negates possible microenvironmental after removal of dye-containing medium and rapid washes with dye-free effects on the excitation and emission spectra and fluorescence medium. In a typical 6-h uptake experiment, forty 60-mm tissue culture intensity. The fluorescence intensity of Rh-123 in butanol was dishes (Falcon, Cockeysville, MD) were evenly seeded with cells and linear with its concentration over the range measured in these grown to subconfluence. Prior to the experiment, 18 of the dishes were experiments (0-150 ng/ml). The excitation and emission maxima aspirated free of medium, wrapped in plastic, and placed at -20°C for were 512 and 532 nm, respectively; these values did not shift 12-16 h. On the day of the experiment, these control dishes were significantly over this concentration range. allowed to thaw at room temperature for 10 min and then each received 3 ml of freshly prepared
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