[CANCER RESEARCH 50, 4167-4172, July 1, 1990] Rhodamine 123 Phototoxicity in Laser-irradiated MGH-U1 Human Carcinoma Cells Studied in Vitro by Electron Microscopy and Confocal Laser Scanning Microscopy1 Christopher R. Shea,2 Margaret E. Sherwood, Thomas J. Flotte, Norah Chen, Manfred Scholz, and Tayyaba Hasan Wellman Laboratories of Photomedicine, Department of Dermatology, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02114 ABSTRACT Rhodamine 123 (R123) is a permeant, cationic, fluorescent dye that localizes preferentially within mitochondria of living carcinoma cells. MGH-U1 human bladder carcinoma cells incubated in vitro with 10 ¿IM R123 for 30 min and then irradiated at 514.5 nm with an argon ion laser underwent selective, phototoxic injury to mitochondria. Ultrastructurally, treatment with R123 plus irradiation with 10 ,1/ciir caused selective, progressive mitochondria! alterations consisting of disruption of cristae, vacuolization, swelling, increasing numbers of ring-shaped and angulated mitochondria at 4 to 8 h after irradiation, and obliteration of many mitochondria at 24 to 48 h. Confocal laser scanning microscopy after treatment with R123 plus irradiation with 10 to 30 J/air demonstrated altered uptake and localization of subsequently administered R123, ac companied by striking mitochondria! fragmentation. Irradiation caused a dose-dependent depletion of extractable R123, due to a photosensitized efflux that began immediately and progressed by 4 h after irradiation with 10 to 30 J/cm2; further uptake after reincubation in the presence of R123 was also quantitatively impaired in cells previously irradiated with 30 J/cm2. INTRODUCTION R1233 is the prototype of a group of permeant, cationic dyes that have been widely investigated in recent years, both as fluorescent probes and as potential agents for chemotherapy of cancer. R123 preferentially localizes in undamaged mitochon dria of living cells (1) largely because of electrophoretic forces generated by the proton gradient across the mitochondrial inner membrane (2). When mitochondria are injured and this gra dient disturbed, R123 assumes a diffuse distribution in the cytoplasm (3). Many types of carcinoma cells in vitro reportedly have an increased avidity for R123 (4) because of an increased electrical potential across the mitochondrial inner membrane (2,5). Incubation in the presence of R123 at high concentrations or for long periods causes mitochondrial toxicity (6-11), lead ing to selective killing of certain carcinoma cells versus non- transformed epithelial cells in vitro (12). R123 chemotherapy of cancer has been studied in vivo in rodent models (13, 14), but toxic effects on normal organs limit its utility as monother- apy, even though there is a significantly greater uptake and retention of R123 in experimental tumors than in normal tissues (14, IS). Combined treatment with R123 and visible-light irradiation is phototoxic to cancer cells in vitro (16-23); photochemother- apy with low-dose R123 might therefore be an effective local modality without severe systemic toxicity (24). R123 in cells Received 10/3/89; revised 2/2/90. 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. 1This work was sponsored by Office of Naval Research Contract NOOO14-86- K-0117; American Cancer Society Grant IN-173; NIH Grant GMA-1, 2 ROI- AR25395; and Arthur O. and Gullan M. Wellman Foundation. 2To whom requests for reprints should be addressed. 3The abbreviations used are: R123, rhodamine 123; CLSM, confocal laser scanning microscopy; DOTC, doxycycline; DPBS. Dulbecco's phosphate-buffered saline; EM, electron microscopy; 'O2, singlet oxygen; TC, tetracycline. absorbs light efficiently at 514.5 nm (16), a wavelength whose optical penetration into tissue is sufficient, and is even consid ered optimal, for treatment of superficial malignancies such as carcinoma in situ of the urinary bladder (25). In vitro, R123 phototoxicity causes significant inhibition of colony formation (16), proliferation (17), and uptake of tritiated thymidine (18) of human bladder carcinoma cells at R123 concentrations and radiant exposures that have no such effects when administered independently. The chemical and biological mechanisms of phototoxicity of R123 are unclear, including the primary pho tochemistry responsible, the cellular lesions produced, and the time course and functional consequences of photosensitized injury. In order to elucidate the details of the mechanisms of R123 phototoxicity, we have assessed the structural alterations of carcinoma cells by transmission EM and CLSM at various intervals after treatment in vitro with R123 followed by argon ion laser irradiation at 514.5 nm. Morphological alterations have been correlated with functional injury to mitochondria, as reflected by a reduced ability of irradiated cells to retain R123 and to concentrate it upon subsequent reincubation in its pres ence. MATERIALS AND METHODS Cells. MGH-U1 cells (26), derived from a human transitional cell carcinoma of the urinary bladder, were grown as subconfluent mono- layers on glass coverslips in McCoy's Medium 5A with 25 ITIM4-(2- hydroxyethyl)-l-piperazineethanesulfonic acid buffer (Gibco Labora tories, Grand Island, NY) supplemented with heat-inactivated 5% fetal bovine serum (Gibco); incubation was at 37°Cin a humidified 95% air:5% CO2 atmosphere. Cells in exponential growth were used for all experiments. Routine cultures for Mycoplasma contamination were consistently negative. Radiation Source. The 514.5-nm emission from an argon ion laser (Model Innova 100; Coherent, Inc., Palo Alto, CA) was directed to the cell monolayer by a fiber optic system at an irradiance of 100 mW/cm2 as previously described (16). No detectable heating occurred at this irradiance. Photosensitization and Irradiation Protocol. Medium was aspirated from cultures and replaced with 10 pM R123 (Eastman Kodak Co., Rochester, NY) in DPBS (Gibco) containing 0.49 mM MgCl2 H2O and 0.9 mM CaCl2 at pH 7.2. After incubation at 37°Cfor 30 min in the dark, cultures were washed twice in R123-free DPBS, immediately irradiated (3, 10, or 30 J/cm2) while in DPBS, and then either imme diately fixed for EM, subjected to extraction in «-butylalcohol, or covered with R123-free medium and incubated until ready for fixation, extraction, or viewing by CLSM. Control experiments were performed in parallel, with cultures exposed to R123-free DPBS with or without irradiation, or to R123 without irradiation. The radiant exposure range used has been shown previously to cause dose-dependent phototoxicity to MGH-U1 cells only after treatment with R123 (16-18). Electron Microscopy. Cells were washed twice in DPBS, fixed in 4% glutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated in a graded ethanol series, and embedded by inversion of Beem capsules containing Epon 812. Thin sections were stained with uranyl acetate and lead citrate and viewed with an electron microscope (Model CM 10; Philips, Inc., Eindhoven, The Netherlands). Electron micrographs 4167 Research. on November 6, 2021. © 1990 American Association for Cancer cancerres.aacrjournals.org Downloaded from