Rhodamine 123 Phototoxicity in Laser-Irradiated MGH-U1 Human Carcinoma Cells Studied in Vitro by Electron Microscopy and Confocal Laser Scanning Microscopy1

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

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. RHODAMINE 123 PHOTOTOXICITY were taken of cultures fixed immediately and 1, 4, 8, 24, or 48 h after irradiation most mitochondria were moderately swollen, with treatment. disrupted cristae (Fig. id); many mitochondria showed unusual Confocal Laser Scanning Microscopy. At 1, 2, 4, or 26 h after configurations, including ring-shaped and angulated forms with treatment, cells were incubated a second time in R123 solution (10 ^M variably thinned diameters. Other adjacent organdÃes were for 30 min), washed, mounted on glass slides with supplemental me dium without phenol red, and then viewed by CLSM (Wild-Leitz, Inc., entirely normal. At 8 h after irradiation there were more lipid Wetzlar, Federal Republic of Germany) operated in the fluorescence droplets and increased distortion of mitochondria with many mode, with a xlOO, 1.32-numerical aperture, oil immersion objective, bizarre forms (Fig. \e). At 24 and 48 h after irradiation, many using argon ion laser excitation at 488 nm and emission >515 nm. mitochondria were obliterated (Fig. I/), and most of the re Scanning over a smaller field size permitted higher magnification maining mitochondria were swollen, had undergone severe images to be taken (zoom mode). Images were stored on an optical disc disruption or loss of cristae, and contained dense deposits in and processed with a computer system equipped with image enhance the matrix. Cytoplasmic vacuoles (some containing amorphous ment and coloring capabilities. Extended focus images were processed material) and myelin figures were prevalent, and there was from optical sections taken serially at z-plane increments of approxi slight swelling of the Golgi apparatus. The nucleus appeared mately 0.5 litn (27). All CLSM experiments on any particular prepa normal. ration were completed within 15 min, to avoid artifactual changes in Confocal Laser Scanning Microscopy. Living cells treated with cellular morphology and fluorescence. Preliminary experiments showed that repeated scanning caused no detectable photobleaching or photo- R123 alone, in the absence of laser irradiation, displayed bril toxicity under the conditions used. liant fluorescence exclusively within their mitochondria (Fig. Dye Extraction and Quantification. Subconfluent cultures in plastic 2); extramitochondrial sites (e.g., nucleus, cytoplasm) in unir- Petri dishes were incubated with 10 MMR123 for 30 min, washed twice radiated cells were free of R123 fluorescence. Serial optical in DPBS, covered with DPBS, irradiated (10 or 30 J/cm2) or kept in sectioning showed that the mitochondria comprised an inter the dark, treated briefly with 0.05% trypsin and 0.53 HIM EDTA connecting system with multiple loops and branch points. In (Gibco), vigorously pipetted to obtain single cell suspensions, centri- typical optical sections, the apparent length of mitochondria fuged, resuspended, counted (Model ZF; Coulter Electronics, Inc., was about 5 Â¡j.m.Byextended-focus processing of serial optical Hialeah, FL), and immersed in fluorometric grade n-butyl alcohol. sections, composite images were generated in which the overall Extraction was performed either immediately or 4 h after the irradiation period; in additional experiments cells were reincubated with 10 ;<\i spatial continuity of mitochondria was apparent. R123 for 30 min at 4 h after the irradiation period and then immediately R123 phototoxicity caused striking CLSM findings (Fig. 3), subjected to the extraction procedures. Cellular R123 content was namely, an altered localization of R123 (variably within the calculated by comparison of the absorbance of extracts with standard cytoplasm, nuclear membrane, and mitochondria) and struc solutions using a diode array spectrophotometer (Model 8451 A; Hew tural alterations of mitochondria themselves. Most mitochon lett-Packard Co., Sunnydale, CA). Significance of differences in R123 dria appeared fragmented and short (~1 ^m); some were swol content as a function of radiant exposure and time after irradiation was len, globular, or ring-shaped. In general the order of severity of assessed by nonparametric tests (Kruskal-Wallis for comparison of 3 injury was 30 > 10 > 3 ~ 0 J/cm2, and 26 > 4 > 2 ~ 1 h, as groups and Whitney-Mann for paired comparisons); P values < 0.05 judged by qualitative morphological criteria. Phototoxic alter were considered statistically significant. ations were consistent over the course of multiple experiments, with some regional variations in fluorescence intensity and RESULTS mitochondrial morphology. Irradiation caused no detectable alterations of cells not pretreated with R123. Electron Microscopy. Control cells (no R123, no irradiation) Iutrare-Millar R123 Content. Unirradiated cells showed a sig had smooth, rounded plasma membranes with occasional, short nificant decrease in R123 content by 4 h after being covered pseudopodia. Nuclei were eccentrically located and had in with R123-free medium (Fig. 4). Laser irradiation caused a dented contours and peripheral aggregation of chromatin. Or- significantly greater decrease in R123 content, both immedi ganelles, especially Golgi apparatus and mitochondria, were ately after irradiation and at 4 h (P = 0.0001). The total R123 well developed and mainly located next to the nucleus. Most content also varied with radiant exposure in cells reincubated mitochondria were elongated and had well-formed, evenly with R123 at 4 h after the irradiation period (P = 0.0001). spaced cristae (Fig. la). Cytoplasmic vacuoles, lipid droplets, After reincubation, however, unirradiated cells and cells previ ously irradiated with 10 J/cm2 showed an identical net increase and myelin figures were rarely observed. Cells fixed immedi ately or l h after irradiation with 10 J/cm2 alone, in the absence in R123 content (mean change in content = 1.2 x 10~16mol/ of R123, appeared similar to control cells; those fixed from 4 cell); that is, net reuptake of R123 at this time was unaffected by previous irradiation with 10 J/cm2. Cells previously irradi to 48 h after irradiation showed slightly increased vacuolization ated with 30 J/cm2, on the other hand, showed both a signifi in the cytoplasm and slight disruption or loss of cristae. In cantly impaired reuptake of R123 (mean change in content = general, the alterations caused by treatment with R123 alone, 7.2 x 10~17mol/cell, P < 0.02) and a low total R123 content without irradiation, were subtle and did not vary with time after treatment. Mitochondria of cells treated with R123 alone were after reincubation with R123 (P = 0.0001), compared with predominantly elongated (Fig. 1Â¿>);some were rounded and unirradiated cells. swollen or had focally disrupted cristae resulting in internal vacuolization. Cells treated with R123 alone also contained a DISCUSSION greater number of cytoplasmic vacuoles, myelin figures, and lipid droplets compared with control cells. R123 is a useful biological probe because of its high fluores Combined treatment with R123 and irradiation with 10 J/ cence efficiency (28), relatively low toxicity when used at low cm2 caused striking mitochondria! alterations, which pro concentration for brief periods, and selective affinity for mito gressed over time. Immediately after irradiation no changes chondria of living cells, in particular carcinoma cells. The were identified beyond the focal disruption of cristae seen in pattern and intensity of R123 fluorescence vary with the elec cells treated with R123 alone (Fig. le). At l h after irradiation trical potential gradients across the mitochondrial inner mem some mitochondria appeared more distorted. At 4 h after brane and the plasma membrane (2), cell type (4), species (29), 4168

'Â«â€¢Â«:'â€¢'-,'"1â€¢â€¢'

Fig. 1. Transmission electron micros copy demonstrates the ultrastructural ef fects of R123-sensitized photochemical re actions after irradiation at S14.S nm. In a, control cells (no R123, no irradiation) ex hibit elongated mitochondria with evenly spaced cristae and a uniformly distributed, electron-dense matrix. In b, cells fixed im mediately after treatment for 30 min with 10 MMR123 alone (no irradiation) exhibit well-formed mitochondria with minor in ternal vacuolization (arrows). The number of lipid droplets, myelin figures, and cytoplasmic vacuoles is increased, compared with control cells. In c. cells pretreated with 10 /Â¡MRI23 for 30 min and fixed immediately after irradiation with 10 J/ cm2 exhibit mitochondria with some dis ruption of cristae (arrows), similar to those in cells treated with R123 alone (b). In d, at 4 h after irradiation with 10 .I/cm2, cells pretreated with 10 UM R123 for 30 min show markedly swollen mitochondria with disrupted cristae. Some mitochondria ex hibit unusual configurations, predomi nantly ring forms and angulated forms (ar rows). In e, at 8 h after irradiation with 10 J/cm2, mitochondria of cells pretreated with 10 MMR123 for 30 min show in creased distortion. Very few normal ap pearing mitochondria are present. There is also increased lipid formation (asterisks). In f, at 48 h after irradiation with 10 J/ cm2, most mitochondria in cells pretreated with 10 MMfor 30 min are obliterated. Those mitochondria observed are swollen and show almost total loss of cristae (arrows). Dense deposits are observed in the mitochondria. Myelin figures are prevalent (asterisks), and moderate swelling of the Golgi apparatus is seen. (Bar = 1 ^m for all electron micrographs.)

phase of the cell cycle (30), rate of proliferation (31), growth exploits both this property of R123 and its ability to sensitize and differentiation (32), and metabolic status and health of the phototoxic reactions within mitochondria selectively. cell (33). For these reasons, R123 can be used as a probe of R123 treatment in the absence of irradiation reportedly can both structure and function of mitochondria. The present study alter mitochondrial ultrastructure (34, 35), but in our study the 4169

2 - UJ u

ce aLU

4 hr, then Ohr 4hr re-incubate with R123 TIME POST IRRADIATION Fig. 4. Intracellular R123 content as a function of radiant exposure. Cells were pretreated with R123 (10 ^M for 30 min) and either shielded from light or irradiated at 514.5 nm. D, unirradiated cultures;E3, cultures irradiated with 10 J/ cm2; â€¢.cultures irradiated with 30 J/cm2. There is a significant dose-dependent and time-dependent decrease in R123 content, and the R123 content of cells reincubated with R123 at 4 h after irradiation also varies with dose (radiant exposure). Columns, mean of 9 to 18 cultures at each entry; bars, SE.

striking alterations by treatment with R123 alone generally Fig. 2. High-magnification (zoom 4) CLSM of a perinuclear region of a single, requires higher ambient R123 concentrations or longer incu unfixed cell treated with 10 IÂ¿MR123for 30 min without irradiation. Extended bation times, which lead to higher intracellular R123 content focus composite image taken from 10 single scans demonstrates the complex, largely continuous mitochondria! system. The intensity of fluorescence is propor and increased toxicity (34). LI210 cells treated with sufficient tional to color (white > yellow > red > black). Note that the nucleus (in the lower R123 in the absence of irradiation reportedly show intracristal left corner) and intermitochondrial cytoplasmic spaces do not fluoresce. (Bar = 5 vacuolization and distortion, an increase in the number of (im). matrix granules, and ring-shaped mitochondria (35). In the present study, very similar findings were prominent in MGH- Ul cells treated with low-dose R123 plus 10 J/cm2 laser light. Laser irradiation thus appears to cause a true potentiation of the intrinsic toxicity of R123, even inducing similar ultrastruc tural alterations. Ring-shaped mitochondria (which are rarely seen in normal cells) are probably a consequence of mitochon dria! fragmentation; they can be induced by other mitochondrial toxins as well (36). The ultrastructural changes induced in mitochondria by R123 phototoxicity differ greatly from those seen in cells treated with the phototoxic drug DOTC and then irradiated with ultraviolet A, 320-400 nm (37); the differences are notable because DOTC, like other TCs (38), is also concentrated selectively within mitochondria and sensitizes mitochondrion-specific phototox icity. DOTC phototoxicity causes mitochondria to swell mark edly (up to 5 times their normal diameter) and to lose the ability to concentrate R123 (37, 39); these responses begin by 10 min after irradiation, are maximal by 1 h, and are partially reversed by 4 h. In contrast, R123 phototoxicity causes mitochondrial alterations that are most evident many hours after irradiation and are not reversed by 48 h. Furthermore, the phototoxic swelling induced with R123 is not as massive as with DOTC; rather, R123 phototoxicity primarily causes the mitochondria to undergo fragmentation and bizarre changes in configuration. Fig. 3. Normal magnification, extended focus CLSM of unfixed cells 4 h after These different results probably reflect differences in both pri R123 pretreatment (10 ^M for 30 min) and irradiation with 10 J/cm2 at 514.5 mary photochemistry responsible for injury and the specific nm. Mitochondria are markedly fragmented but tend to remain brightly fluores suborganelle site of injury. TC phototoxicity is largely oxygen- cent. A background of diffuse fluorescence, including localization in the nuclear membrane, is apparent. (Bar = dependent (40), and the generation of 'O2 is a major phototoxic mechanism (39-41). 'O2 efficiently induces membrane oxida changes induced by R 123 alone were rather subtle; this finding tion (42), leading to increased permeability and swelling, gen is consistent with the lack, in our system, of any detectable erally within a few minutes after irradiation (43). In contrast, effects of R 123 alone on colony formation, thymidine incor R123 phototoxicity is progressively manifested at much later poration, proliferation, or vital staining (16-18), probably be times after irradiation, consistent with the low (<1%) quantum cause of the relatively low intracellular R 123 concentration yield of 'O2 from R123 photoreactions (16) due to the low attained under these incubation conditions. Induction of more efficiency of intersystem crossing to the triplet manifold (28, 4170

44). The biochemical lesion(s) produced by R123 photosensi- uptake presumably results mainly from the electrical potential tization must be investigated further; one plausible target is the across the plasma membrane, which appears intact even at this F0F, ATPase of the mitochondria! inner membrane, a major rather high radiant exposure (16). The altered localization of site of action of R123 in the absence of irradiation (5). R123 after laser irradiation may be a consequence of nonspe The high spatial resolution of CLSM images is a consequence cific impairment of the mitochondrial inner membrane poten of the ability to perform optical sectioning through the z-plane, tial or perhaps photosensitized alteration of specific binding by suppression of out-of-focus optical planes (27, 45). In our sites of R123. study, continuity and interconnections of the unirradiated In summary, R123 photosensitization causes injury selec MGH-U1 cell mitochondria are apparent, a form of mitochon- tively to mitochondria of cultured carcinoma cells, because of drial organization seen in only a few cell types such as mouse toxic photochemical reactions at its site of preferential localiza 3T6 cells (1). In contrast, the MGH-U1 cell mitochondria are tion. This organelle-specific phototoxic injury, with its distinc fragmented after R123 photosensitization; furthermore, R123 tive morphological and functional consequences, is of interest is present in the nuclear membrane of irradiated cells only, for basic studies of photobiology and bioenergetics and may reflecting a redistribution in response to mitochondrial injury. lead to the rational synthesis of new photosensitizers and to Photobleaching of R123 with CLSM in this study is less than further use of tumor mitochondria as targets of novel forms of with conventional epifluorescence microscopy, because the in photochemotherapy. stant storage of images obviates the need for prolonged illumi nation during viewing or photography. Thus the images ob ACKNOWLEDGMENTS tained are stable, and no correction needs to be made for dynamic changes such as fluorescence redistribution in this We thank Dr. Chi-Wei Lin for his gift of MGH-U1 cells, Dr. Reiner study. Peters for help in the CLSM studies, Dr. Sewon Kang for statistical The uptake and efflux of R123, demonstrated qualitatively consultation, Dr. Victor Tron and Dr. Randy Byers for helpful discus by CLSM, have been quantified by extraction. At 4 h after sions and criticisms, Wild-Leitz for the loan of CLSM equipment, and being covered with R123-free medium, unirradiated cells have Gabriele Vogt for expert secretarial help. lost about half the R123 initially present (Fig. 4); the rate of efflux is very similar to that demonstrated in other carcinoma cell lines (46). After a 30-min reincubation in 10 fiM R123 REFERENCES solution, unirradiated MGH-U1 cells take up the same incre 1. Johnson, L. V., Walsh, M. L., and Chen, L. B. Localization of mitochondria mental amount of R123 as they did after the original incubation, in living cells with rhodamine 123. Proc. Nati. Acad. Sci. USA, 77: 990- indicating that mitochondrial binding sites of R123 (47) are 994, 1980. 2. Davis, S., Weiss, M. J., Wong, J. R., Lampidis, T. J., and Chen, L. B. not saturated; in fact, R123 remains selectively localized to Mitochondrial and plasma membrane potentials cause unusual accumulation mitochondria of unirradiated cells even at a total content >1 x and retention of rhodamine 123 by human breast adenocarcinoma-derived 10~15mol/cell (achieved by incubating cells for 60 min in 100 MCF-7 cells J. Biol. Chem., 260: 13844-13850, 1985. 3. Lampidis, T. J., Bernal, S. D., Summerhayes, I. C., and Chen, L. B. Selective fiM R123 solution).4 toxicity of rhodamine 123 in carcinoma cells in vitro. Cancer Res., 43: 717- Laser irradiation of cells markedly alters their R123 content. 720, 1983. Within minutes after irradiation with 10 J/cm2 and 30 J/cm2, 4. Nadakavukaren, K. K., Nadakavukaren, J. J., and Chen, L. B. Increased rhodamine 123 uptake by carcinoma cells. Cancer Res., 45: 6093-6099, the mean R123 content drops to approximately 50% and 27%, 1985. 5. Modica-Napolitano, J. S., and Aprille, J. R. Basis for the selective cytotox- respectively, of that of unirradiated cells. This immediate pho icity of rhodamine 123. Cancer Res., 47: 4361-4365, 1987. tosensitized efflux is easily detected even though injury is not 6. Modica-Napolitano, J. S., Weiss, M. J., Chen, L. B., and Aprille, J. R. yet apparent by EM; thus R123 content is a very sensitive Rhodamine 123 inhibits bioenergetic function in isolated rat mitochondria. Biochem. Biophys. Res. Commun., 118: 717-723, 1984. measure of functional injury to mitochondria. Photodegrada 7. Singh, G., and Shaughnessy, S. G. Functional impairment induced by lipo- tion of R123 probably contributes little to this immediate philic cationic compounds on mitochondria. Can. J. Physiol. Pharmacol., decrease, for only 10% photodegradation occurs in R123 solu 6(5:243-245, 1988. tion irradiated with 40 J/cm2 (17). R123 continues to efflux 8. Abou-Khalil, W. H., Arimura, G. K., Yunis, A. A., and Abou-Khalil, S. Inhibition by rhodamine 123 of protein synthesis in mitochondria of normal from cells by 4 h after irradiation, at a rate directly proportional and cancer tissues. Biochem. Biophys. Res. Commun., 137: 759-765, 1986. 9. Vargas, J. L., Roche, E., Knecht, E., Aniento, F., and Grisolla, S. The to the radiant exposure (Fig. 2); conversely, the total R123 mitochondrial probe rhodamine 123 inhibits in isolated hepatocytes the content of cells incubated a second time in R123 solution after degradation of short-lived proteins. FEBS Lett., 233: 259-262, 1988. 4 h is inversely related to the radiant exposure. Paradoxically, 10. Morita, T., Mori, M., Ikeda, F., and Tatibana, M. Transport of carbamoyl cells previously treated with R123 plus 10 J/cm2 irradiation phosphate synthetase I and ornithine transcarbamylase into mitochondria. J. Biol. Chem., 257: 10547-10550, 1982. take up a normal incremental amount of R123 after 4 h. CLSM 11. Lubin, I. M., Wu, L. N. Y., Wuthier, R. E., and Fisher, R. R. Rhodamine 123 inhibits import of rat liver mitochondrial transhydrogenase. Biochem. shows, however, that the R123 is abnormally distributed, par Biophys. Res. Commun., 144: 477-483, 1987. tially within altered mitochondria and partially within the cy- 12. Bernal, S. D., Lampidis, T. J., Summerhayes, I. C., and Chen, L. B. Rhoda- toplasmic compartment and nuclear membrane. Thus, bulk mine-123 selectively reduces clonal growth of carcinoma cells in vitro. Science (Wash. DC), 218: 1117-1118, 1982. uptake of R123 cannot be the sole criterion of mitochondrial 13. Bernai, S. D., Lampidis, T. J., Mclsaac, R. M., and Chen, L. B. Anticarci injury but must be correlated with other morphological and noma activity in vivoof rhodamine-123, a mitochondrial-specific dye. Science functional data. [For example, we have reported elsewhere that (Wash. DC), 222: 169-172, 1984. R123 pretreatment plus irradiation with 10 J/cm2 causes almost 14. Herr, H. W., Huffman, J. L., Huryk, R., Heston, W. D. W., Melamed, M. R., and Whitmore, W. F., Jr. Anticarcinoma activity of rhodamine 123 total inhibition of cellular proliferation for the first 48 h after against a murine renal adenocarcinoma. Cancer Res., 48: 2061-2063, 1988. 15. Beckman, W. C., Jr., Powers, S. K., Brown, J. T., Gillespie, G. Y., Bigner, irradiation (17).] Compared with unirradiated cells, cells irra D. D., and Camps, J. L., Jr. Differential retention of rhodamine 123 by avian diated with 30 J/cm2 take up a significantly lower incremental sarcoma virus-induced glioma and normal brain tissue of the rat in vivo. amount of R123 upon reincubation; CLSM shows that this Cancer (Phila.), 59: 266-270, 1987. 16. Shea, C. R., Chen, N., Wimberly, J., and Hasan, T. Rhodamine dyes as R123 is localized mainly in the cytoplasm, and therefore its potential agents for photochemotherapy of cancer in human bladder carci noma cells. Cancer Res., 49: 3961-3965, 1989. * C. R. Shea et al., unpublished data. 17. Shea, C. R., Chen, N., and Hasan, T. Dynamic aspects of rhodamine dye 4171

photosensitization in vitro using an argon-ion laser. Lasers Surg. Med., 9: 33. Evenson, D. P.. Darzynkiewicz, Z., and Melamed, M. R. Simultaneous 83-89, 1989. measurement by flow cytometry of sperm cell viability and mitochondrial 18. Shea, C. R., Chen, N., and Hasan, T. Phototoxicity of rhodamine dyes. membrane potential related to cell motility. J. Histochem. Cytochem., 30: Proc. Soc. Photo-optical Instrum. Eng., 997; 48-57, 1988. 279-280, 1982. 19. Powers, S. K., Pribil, S., Gillespie, G. Y., and Watkins, P. J. Laser photo- 34. Tapiero, H., Sbarbati, A., Fourcade, A., Cinti, S., and Lampidis, T. J. Effect chemotherapy of rhodamine-123 sensitized human glioma cells in vitro. J. of verapamil on rhodamine 123 mitochondrial damage in Adriamycin resist Neurosurg., 64: 918-923, 1986. ant cells. Anticancer Res., 6: 1073-1076, 1986. 20. Oseroff, A. R., Ohuoha, D., Ã„ra,G., McAuliffe, D., Foley, J., and Cincotta, 35. Evenson, D. P., Lee, J., Darzynkiewicz, Z., and Melamed, M. R. Rhodamine L. Intramitochondrial dyes allow selective in vitro photolysis of carcinoma 123 alters the mitochondrial ultrastructure of cultured LI210 cells. J. His cells. Proc. Nati. Acad. Sci. USA, 83: 9729-9733, 1986. tochem. Cytochem., 33: 353-359, 1985. 21. Castro, D. J., Saxton, R. E., Fetterman, H. R., Castro, D. J., and Ward, P. 36. Kass, M. M. K. Analysis of methylglyoxal bis(guanylhydrazone)-induced H. Rhodamine-123 as a new photochemosensitizing agent with the argon alterations of hamster tumor mitochondria by correlated studies of selective laser: "nonthermal" and thermal effects on human squamous carcinoma cells rhodamine binding, ultrastructural damage, DNA replication, and reversibil in vitro. Laryngoscope, 97: 554-561, 1987. ity. Cancer Res., 44: 2677-2688, 1984. 22. Melloni, E., Dasdia, T., Fava, G., Rocca, E., Zunino, F., and Marchesini, R. 37. Shea, C. R., Whitaker, D., Murphy, G. F., and Hasan, T. Ultrastructure and In vitro photosensitizing properties of rhodamine 123 on different human dynamics of selective mitochondrial injury in carcinoma cells after doxycy- tumor cell lines. Photochem. Photobiol., 48: 311-314, 1988. cline photosensitization in vitro. Am. J. Pathol., 133: 381-388, 1988. 23. Darzynkiewicz, Z., and Carter, S. P. Photosensitizing effects of the tricyclic 38. DuBuy, H. G., and Showacre, J. L. Selective localization of tetracycline in heteroaromatic cationic dyes pyronin Y and toluidine blue O (tolonium mitochondria of living cells. Science (Wash. DC), 133: 196-197, 1961. chloride). Cancer Res., 48: 1295-1299, 1986. 39. Shea, C. R., Wimberly, J., and Hasan, T. Mitochondrial phototoxicity 24. Powers, S. K., Beckman, W. C, Brown, J. T., and Kolpack, L. C. Interstitial sensitized by doxycycline in cultured human carcinoma cells. J. Invest. laser photochemotherapy of rhodamine-123-sensitized rat glioma. J. Neu Dermatol., 87: 338-342, 1986. rosurg., 67: 889-894, 1987. 40. Hasan, T., Kochevar, I. E., McAuliffe, D. J., Cooperman, B. S., and Abdulah, 25. Nseyo, U. O., Whalen, R. K., Duncan, M. R., Berman, B., and Lundahl, S. D. Mechanism of tetracycline phototoxicity. J. Invest. Dermatol., 83: 179- Immune response following photodynamic therapy for bladder cancer. Proc. 183, 1984. Soc. Photo-optical Instrum. Eng., 1065: 66-72, 1989. 41. Hasan, T., and Khan, A. U. Phototoxicity of the tetracyclines: photosensi 26. Lin, C-VV.,Lin, J. C., and Prout, G. R. Establishment and characterization tized emission of singlet delta dioxygen. Proc. Nati. Acad. Sci. USA, 83: of four human bladder tumor cell lines and sublines with different degrees of 4604-4606, 1986. malignancy. Cancer Res., 45: 5070-5079, 1985. 42. Valenzo, D. P. Photomodification of biological membranes with emphasis 27. Scholz, M., Sauer, H., Rihs, H.-P., and Peters, R. Laser scanning microscopy on singlet oxygen mechanisms. Photochem. Photobiol., 46: 147-160, 1987. to study molecular transport in single cells. Proc. Soc. Photo-optical Instrum. 43. Moan, J., Pettersen, E. O., and Christensen, T. The mechanism of photo- Eng., 702Â«:160-166, 1988. dynamic inactivation of human cells in vitro in the presence of haematopor- 28. Kubin, R. F., and Fletcher, A. N. Fluorescence quantum yields of some phyrin. Br. J. Cancer, 39: 398-407, 1979. rhodamine dyes. J. Lumin., 27: 455-462, 1982. 44. Chow, A., Kennedy, S., Pottier, R., and Truscott, T. G. Rhodamine 123â€” 29. Gupta, R. S., and Dudani, A. K. Species-specific differences in the toxicity photophysical and photochemical properties. Photobiochem. Photobiophys., of rhodamine 123 toward cultured mammalian cells. J. Cell Physiol., 130: /;.â€¢139-148, 1986. 321-327, 1987. 45. Brakenhoff, G. J., van der Voort, H. T., van Spronsen, E. A., and Nanninga, 30. Benel, L., Ronot, X., Kornprobst, M., Adolphe, M., and Mounolou, J. C. N. Three-dimensional imaging by eonfocal scanning fluorescence micros Mitochondria! uptake of rhodamine 123 by rabbit articular chondrocytes. copy. Ann. NY Acad. Sci., 483: 405-415, 1986. Cytometry, 7: 281-285, 1986. 46. Summerhayes, I. C., Lampidis, T. J., Bernai, S. D., Nadakavukaren, K. K., 31. Darzynkiewicz, Z., TrÃ¡ganos, F., Staiano-Coico, L., Kapuscinski, J., and Shepherd, E. L., and Chen, L. B. Unusual retention of rhodamine 123 by Melamed, M. R. Interactions of rhodamine 123 with living cells studied by mitochondria in muscle carcinoma cells. Proc. Nati. Acad. Sci. USA, 79: flow cytometry. Cancer Res., 42: 799-806, 1982. 5292-5296, 1982. 32. Collins, J. M., and Foster, K. A. Differentiation of promyelocytic (HL-60) 47. Bunting, J. R., Kamali, E., Phan, T. V., Dowben. R. M., and Matthews, J. cells into mature granulocytes: mitochondrial-specific rhodamine 123 fluo L. Study of the specificity of xanthene dye binding to mitochondria. Proc. rescence. J. Cell Biol., 96: 94-99, 1983. Soc. Photo-optical Instrum. Eng., 997: 58-61. 1988.

4172

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. Rhodamine 123 Phototoxicity in Laser-irradiated MGH-U1 Human Carcinoma Cells Studied in Vitro by Electron Microscopy and Confocal Laser Scanning Microscopy

Christopher R. Shea, Margaret E. Sherwood, Thomas J. Flotte, et al.

Cancer Res 1990;50:4167-4172.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/50/13/4167

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/50/13/4167. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.