Med Electron Microsc (2002) 35:53–59

© The Clinical Electron Microscopy Society of Japan 2002

ORIGINAL PAPER Jojiro Nakada · Hiroyuki Ito · Nozomu Furuta Yukihiko Oishi · Yutaka Yamaguchi Tomokazu Matsuura · Yoshiaki Hataba · Seishi Nagamori

Ultrastructure of human pheochromocytoma cells cultured for long periods

Received: September 26, 2001 / Accepted: December 4, 2001

Abstract We conducted ultrastructural analysis of human pheochromocytoma (PC) cells maintained in primary culture for about 10 months. The cells were first isolated by the enzymatic treatment of a surgically resected tissue specimen obtained from a 37-year-old man with PC, a condition which is characterized by elevated blood levels of adrenaline and noradrenaline. It was found that noradrenaline production in the medium continued until the 90th day of culture (1330 pg/ml). The production level decreased to 20 pg/ml on the 180th day, and to 18 pg/ml on the 300th day. Examination under a transmission electron microscope (TEM) at 4 weeks of culture revealed electron-dense granules (about 200 nm in size and, presumably, rich in catecholamines), which were also observed in the tumor cells from the original PC tissue. Neurite-like processes grew at around 1 week of culture, and were still maintained at 6 months of culture. But, after 6 months of culture, the neurite-like processes contained a rosary-like elevated structure, which was suggestive of cell degeneration, as determined by a plasma polymerization replica method and

J. Nakada (*) · H. Ito · N. Furuta · Y. Oishi Department of Urology, Jikei University School of Medicine, 3-25-8 Nishishimbashi, Minato-ku, Tokyo 105-8461, Japan Tel. ⫹81-03-3433-1111, ext. 3561; Fax ⫹81-03-3437-2389 e-mail: [email protected] Y. Yamaguchi Department of Pathology, Jikei University School of Medicine, Tokyo, Japan T. Matsuura Department of Internal Medicine, Jikei University School of Medicine, Tokyo, Japan Y. Hataba Institute of DNA Medicine, Jikei University School of Medicine, Tokyo, Japan S. Nagamori Department of Clinical Pharmacology, Kyorin University School of Medicine, Tokyo, Japan A summary of this paper was presented at the 29th meeting of the Clinical Electron Microscopy Society of Japan, Sapporo, October 1997.

observed with a scanning electron microscope. When cells were examined under the TEM, fewer electron-dense granules were observed in the cell bodies, with more numerous lipofuscin-like granules and filaments. Thus, electron-dense granules, which, presumably, contain catecholamines, were seen in a long-term culture of human PC cells. These granules decreased in number in parallel with the decrease in catecholamine levels in the culture. Key words Human pheochromocytoma · Long-term cell culture · Ultrastructure

Introduction Rat pheochromocytoma (PC) cell lines, for example, PC12, are very useful for studying the mechanism of differentiation and proliferation of nerve cells.1 To date, however, no human-derived PC cell line equivalent to the rat-derived PC12 has been developed. Studies of cellular and functional interactions treated with agents in endocrine pathology have been reported in detail using the methods of electron microscopy.2,3 In studies using cultures of human-derived PC cells, evaluation of the effects of the addition of nerve growth factor or dexamethasone on the catecholamine secretion and cell morphology have been reported.4,5 Reports of long-term primary cultures of human PC cells (about 7 months to 1 year) have also been published.6 In the present study, we attempted the primary culture of human PC cells for about 10 months. The ultrastructure of the cells after long-term incubation was studied by transmission and scanning electron microscopy, and by a combination of threedimenstional transmission electron microscopy and the plasma polymerization replica method (PPRM), which involves glow discharging. The cells used in this study were derived from the patient with PC (case 1) listed in reference 5.

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Subject A 37-year-old male PC patient visited our hospital in February, 1995. His blood adrenaline level (383 pg/ml; normal range, below 100 pg/ml) and noradrenaline level (1309 pg/ml; normal range, 100 to 450 pg/ml) were higher than normal. The blood dopamine level (20 pg/ml) was, however, within the normal range (below 20 pg/ml). The tumor measured about 3 cm in diameter and was resected. Informed consent to study the cell culture using this tumor was obtained from the patient.

Methods The tumor cells were isolated and cultured according to a previously described method.5,7 Briefly, the resected tumor specimen was cut into pieces with scalpels and the pieces were dispersed in Williams’ E medium containing 1000 U/ml dispase (Godo Shusei, Tokyo, Japan). The mixture was incubated for about 1 h to disperse the cells, and was then filtered through a stainless steel mesh. The filtrate was centrifuged to collect a suspension of the isolated cells. The suspension was diluted with Williams’ E medium supplemented with 10% fetal bovine serum (FBS) and inoculated onto a plastic dish coated with type I collagen (Sigma Chemical, St. Louis, MO, USA). The cells were cultured at 37°C in the presence of 5% CO2 and 95% air. The medium was renewed every 2–3 days. For transmission electron microscopy, the cultured cells were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, and postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer. The specimens were then dehydrated in ascending concentrations of ethanol, and embedded in an Epon-Araldite mixture. The ultrathin sections thus obtained were stained with uranyl acetate for examination of the cellular ultrastructure by transmission electron microscopy (Model 1200EX; JEOL, Tokyo, Japan). For scanning electron microscopy, the cultured cells were fixed in 1.2% glutaraldehyde in 0.1 M phosphate buffer, and postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer. Following dehydration, cells were submersed in 100% isoamine acetate for 20 min and then desiccated to critical point under pressure. The cells were then coated with carbon and gold; observations were carried out at 10 KV with a scanning electron microscope (JSM-35; JEOL). For the PPRM, the cultured human PC cells on a plastic dish were treated with phosphate-buffered saline (PBS), fixed in 1.2% glutaraldehyde, and dehydrated in ethanol. The dish was then cut into about 1-cm pieces to prepare plasma polymerization films. The specimens were left to stand on a cathode stage within a low-vacuum tank in a plasma nano-replica apparatus (Model RNR-110; Ushio, Yokohama, Japan). Glow discharging was then performed on the specimens, causing a thin polymerization film of naphthalene gas to form on them. The film-coated plastic

dish pieces containing the specimens were dissolved in propylene oxide, and the insoluble residue was dissolved in sodium hypochlorite. The films were then examined under a transmission electron microscope (TEM).8,9

Results When the time-course of changes in the levels of the three catecholamines in the culture was followed, the levels of adrenaline and noradrenaline were found to by very high on the 1st and 3rd days of incubation. On the 90th day, while the level of adrenaline was found to be below the limit of detection (5 pg/ml), that of noradrenaline remained high, at 1330 pg/ml. Noradrenaline production was detected even on the 120th day (46 pg/ml) and 180th day (20 pg/ml), although at low levels. On the 300th day, noradrenaline production was still detected (18 pg/ml); however, the culture was discontinued at this point.5 When examined under a phase-contrast microscope, viable cells were visualized as dark-colored and round on the 1st day of culture. On the 5th day, the cells assumed a polygonal shape. On the 7th day, several neurite-like processes began to grow from the cells, causing the cells to form a network. On the 180th day, granules with high levels of brightness were visible in the cell body, and the neurite-like processes had formed a more sophisticated network (Fig. 1A,B). When examined under a scanning electron microscope (SEM) on the 180th day of culture, the neurite-like processes extending from the cells were visualized more clearly than under the phase-contrast microscope (Fig. 2A). A rosary-like elevated structure was visible in these processes (Fig. 2B). When specimens prepared by the PPRM were observed under a TEM, the elevated round structure was noted to be adherent to the cell surface, and the rosary-like elevated structure in the neurite-like processes was visualized more clearly than under the SEM (Fig. 3A–D). When the PC tumor specimens were observed under a TEM, the tumor cells were found to contain numerous granules in the cytoplasm. The granules, which ranged in size from 200 to 300 nm, and had a core and high electron density, were distributed diffusely within the cell.5 On the 28th day, cells containing granules and resembling those of the original tissue were seen under a TEM, and the size of these granules (around 200 nm) was about the same as the size of the granules in the cells of the original tissue. These granules, which, presumably, contained catecholamines, had lost their membrane, and had changed into crystal-like, electron-dense granules (Fig. 4A,B). On the 180th day, TEM revealed fewer electron-dense granules, and more numerous lipofuscin-like granules and filaments in the cell body. No membrane was seen around the lipofuscin-like granules. Small dense granules were seen inside the lipofuscin-like granules, and the diameter of these small granules was similar to that of catecholamine granules (Fig. 4C,D).

55 Fig. 1A,B. Phase-contrast micrographs of human pheochromocytoma (PC) cells after 6 months of culture. A Cell body; B neurite-like processes formed a network

Discussion Analysis of the time-course of changes in the catecholamine levels in the culture revealed particularly high levels of adrenaline and noradrenaline on the first and third days of incubation. These findings suggest that cells were destroyed when the specimens were cut into pieces during the preparation of cells for incubation, causing the catecholamines to be released outside the cells.5,10 However, because the culture medium was renewed every 2–3 days, the results of

this study also suggest that catecholamine production continued during the culture and that a significant percentage of the cells remained viable in the long-term culture. Peragner and Walser4 reported that they had succeeded in culturing human PC cells from two patients for long periods of time (7 months to 1 year), but the catecholamine level reported in their study was about 26 pg/ml on the 203rd day. In our study, the noradrenaline level was 1330 pg/ml on the 90th day and 20 pg/ml on the 180th day, more or less identical to the levels reported by Peragner and Walser.6

56 Fig. 2A,B. Scanning electron microscope (SEM) micrographs of human PC cells after 6 months of culture. A Low-magnification micrograph. B Higher magnification SEM micrograph shows the neurite-like processes extending from the human PC cells

We discuss the reason why adrenaline became unmeasurable early in the culture, suggesting that adrenaline may not be produced during culture. Anatomically, the arteriae medullae pass vertically from the subcapsular plexus and straight to the zona reticularis, where they branch off in all directions after distinct spiral twisting. Besides these, the deep capsular arteries from the hilus and from the constricted parts of the glands supply the medulla.11 It had been observed that the ratio of adrenaline to noradrenaline in the adrenal depends on the amount of cortex surrounding the medulla. Species having a high ratio of adrenal cortex to medulla have the highest concentration

of adrenaline and vice versa. These findings suggested that the adrenal cortex had an influence in the synthesis of catecholamines in the adrenal medulla.12 Dexamethasone activates phenylethanolamine-N-methyltransferase (PNMT), an enzyme that converts noradrenaline to adrenaline.13 Indeed, Tischler et al.14 reported that, in cultured chromaffin cells obtained from the adrenal medulla of young adult rats, catecholamine formation, including adrenaline, was maintained on the addition of dexamethasone to the medium. We speculate that, in the present study, adrenaline became unmeasurable early in the culture because the cells were cultured in medium without dexamethasone.

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Fig. 3A–D. Transmission electron microscope (TEM) photographs of human PC cells were taken, using the plasma polymerization replica method (PPRM), after 6 months of culture. A Note cell body. B Highmagnification TEM micrograph of the cell surface of human PC cells.

C TEM micrograph of the neurite-like processes of human PC cells. D TEM micrograph of the rosary-like elevated structure (arrowheads) in the neurite-like processes of human PC cells

When the cultured cells were examined under a phasecontrast microscope, the cells appeared as dark-colored bodies with a small circular form on the first day of culture, but neurite-like processes began to be observed on the seventh day. Similar morphological changes in the cells were also reported by Jaques et al.,15 who, in their study, incubated human PC tissue from 18 patients and examined the cells under a phase-contrast microscope. According to their report, the cells during the early stages of culture were epithelium-like and appeared as dark-colored bodies with a round form. The round cells frequently coalesced into clumps or groups and flattened somewhat as the cell membranes became less distinct. They also reported that these cells changed their morphology to assume a polygonal or stellate form, eventually developing into cells with long axon-like processes. It has been reported that the growth of these neurite-like processes is accelerated following treatment with nerve growth factor (NGF).4–6,10 In the present study, neurite-like processes that caused cross-linking of the cells were seen in the NGF-free cultures

until the 180th day under a phase-contrast microscope, and were still observed on the 300th day. Such processes were observed when the cultures were examined under an SEM on the 180th day. No reports of the observation of neuritelike processes extending from human PC cells examined under an SEM have been published previously. In the present study, we attempted to examine the cultured cells three-dimensionally under a TEM in specimens prepared by a replica method. Yamaguchi et al.8 used this method for the ultrastructural examination of the cell surface of yeast protoplasts, and reported that this method allows highresolution three-dimensional observation and may be more useful than scanning electron microscopy. Sujino9 used a similar method to observe the ultrastructure of cultured liver carcinoma cells and reported that the replica method was useful for observing the surface and cytoskeletal morphology of cultured liver carcinoma cells. In the present study, cells were observed after 6 months of culture, using the replica method. The rosary-like elevated structure on the neurite-like processes could be observed at a higher

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Fig. 4A–D. TEM micrographs of human PC cells. A, B After 4 weeks of culture, electron-dense granules were visible around the nuclei. C, D After 6 months of culture, there was a decrease in the number

resolution than under a phase-contrast microscope or an SEM. It was suggested that the elevated structures were denatured neurite-like processes. Another possibility was that these structures represented a blind-sac-like product of the denaturation of the neurite-like processes, because the structures were contiguous with intact neurite-like processes. TEM examination after 180 days of culture revealed fewer electron-dense granules compared with the number after 4 weeks of culture. Peragner and Walser6 reported that, when cultured human PC cells were observed under a fluorescence microscope, catecholamine fluorescence decreased with time after the start of incubation, but was still detectable (although in trace levels) on the 247th day. Their finding of a steady decrease in the number of catecholamine granules during long-term incubation was endorsed in the present study. In this study, no boundary membrane was seen in the dense granules with a small circular form in the cell body. These irregularly shaped lipofuscin-like granules were often seen in cases of regressive degeneration, and may represent the outcome of fusion between catecholamine granules.

of electron-dense granules, and irregularly shaped lipofuscin-like granules (arrowheads) were observed (D)

Conclusion When human PC cells were cultured for long periods of time, the morphology of the cells and the neurite-like processes extending from the cells changed with time, and these changes were accompanied by a decrease in the number of electron-dense granules, which, presumably, contained catecholamines. The cellular network formed by these neuritelike processes was, however, preserved throughout the long-term period of the culture. Acknowledgments The study was supported in part by a Grant-in-aid for Scientific Research (C) from the Ministry of Education, Science, Sports, and Culture (grant no. 10671496) of Japan.

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