THE ULTRASTRUCTURE OF PIGMENTED MELANOMA CELLS IN CONTINUOUS CULTURE*

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1 THE ULTRASTRUCTURE OF PIGMENTED MELANOMA CELLS IN CONTINUOUS CULTURE* FTJNAN HU, M.D.f AND ROBERT R. CARDELL, JR., Pu.D.t The ultrastructure of mammalian melanocytes (1 7) and melanoma cells (8 17) has been studied by various workers. The fine structures of melanoma cells from different animal sources such as human, mouse, and hamster were found to be similar (16). Melanin granules of normal and malignant melanocytes were found to consist of particulate sub-units of variable form, described as rod-shaped, granular, and lamellated; these sub-units were sometimes noted to exhibit a periodicity of very small dimensions, generally oriented transversely to the long axes of the rods and lamellae. Barnicot and Birbeck (2) were the first to point out that melanocytes contain a system of membranes similar to those seen in the secretory cells. They observed that melanin granules in the cytoplasm of the melanocytes appear to exist in varying stages of development. Seiji et at (15) based on their biochemical and ultrastructural evidence recognized three stages, i.e. pro-tyrosinase, premelanosome and melanosome, in the formation of the melanin granule, the final product of the melanocyte. Moyer (17), in comparing the fine structure of melanosomes in normal and malignant melanocytes of mice carrying various mutant alleles at genetic loci affecting pigmentation, suggested that the melanosomes originated when the protein products of several genetic loci aggregate in the melanocyte cytoplasm to form the protein matrix on which melanin is deposited. In the present report the ultrastructure of the cells of a melanoma cell line (18) was studied. This line was established and propagated in the laboratory of one of us (F. H. ) from a mouse melanoma. MATERIALS AND METHODS A pigmented cell strain, originated from a BiG mouse melanoma and propagated in monolayer cultures, was used exclusively in this study. * From the Department of Dermatology, t Henry Ford Hospital, Detroit, Michigan and the Department of Physics, * Edsel B. Ford Institute for Medical Research, Detroit, Michigan. This study was supported by USPHS Grant CA from the National Cancer Institute. Received for publication June 24, These melanoma cells were grown on glass in medium 199 enriched with fetal bovine serum. When freshly subcultured, the cells were not apparently melanotic. In the course of 4 5 days, isolated colonies of pigmented cells appeared. These gradually increased in size until the glass bottom of the culture bottle became speckled with black spots. At this stage, the cells were lightly trypsinized, rinsed with physiological saline and centrifuged. The pellets were fixed in 1% osmium tetroxide in veronal buffer for 1 hour. Then the pellets were processed through acetone dehydration and embedded in Vestopal W. as described in a previous communication (19). During the process of dehydration the pellets were stained with 2% phosphotungstic acid in absolute alcohol for 1 hour. The specimen was sectioned on an LKB ultratome and the sections placed on parlodian-carboncoated grids. The sections were stained with lead hydroxide after the Karnovsky method (20) and were studied with a modified RCA EMU-2B electron microscope. The cells of a 7th subculture after 8 days of incubation were used exclusively in this study. For light microscopy the cells were examined in living state without staining and fixation. RESULTS Light IlIicroscopy Only two main cell types are recognized, (1) cells filled with melanin granules and, (2) cells without melanin granules. The morphology of these cells varied from small round to ovoid, stellate, bipolar, and multidendritic. The pigment-containing cells often were slightly larger than the non-pigmented cells (Fig. 1). In addition, there was another cell which differed somewhat from the above mentioned cells. The cells were relatively large, epithelial-like cells, and they contained melanin granules which, instead of being evenly distributed in the cytoplasm, were located in the perinuclear position. Electron Illicro.scopy The cells may be arbitrarily divided into three groups. These are: (1) Melanin-filled cells, (2) Light cells, and (3) Dark cells. (1) Melanin-filled cells: These cells were characterized by numerous melanin granules scattered throughout the cytoplasm. Frequently a crosssection of a process was observed and this had

2 68 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY tjh,' I ck *; 1k : V 4ie: A../ r) 4: :t FIG. 1. A phase-contrast micrograph showing the general population of cells. Note the dark pigmented cells and light non-pigmented cells. pre-melanosomes, melanosomes, and mature melanin granules (Fig. 2) in the cytoplasm. (2) Light cells: The light cells were characterized by the pale appearance of the cytoplasm (Fig. 3). The cytoplasm was the most highly vacuolated of the three types of cells. The mitochondria were small and not very numerous. The rough endoplasmic reticulum (ER) appeared as longitudinal sections of tubular units which were encrusted with ribonucleic particles (RNP). Golgi zones were frequently found throughout the cytoplasm. The light cell generally had a smoother outline than the dark cell (Fig. 4). (3) Dark cells: The dark cells had a much denser cytoplasm which was filled with RNP particles (Figs. 3, 4). The dark cells had the largest and most numerous mitochondria of the three groups of cells. The Golgi and ER were not as well developed in the dark cell as in the light cell. The outline of the dark cell was frequently very irregular with many villus-like projections. The fine structure of the nucleus, endoplasmic reticulum, Golgi zone, mitochondria, and melanin granules are similar in these cells, therefore they will be described together. Nucleus The nuclei were irregular in size and shape from cell to cell. The nucleus contained nucleoli and other electron-dense areas. fhc nucleus was enclosed by the nuclear envelope which was composed of the inner and outer nuclear membrane (Fig. 5). Encloplasmic Reticulum (ER) The ER appeared as rough-surfaced tubules and small vesicles (Figs. 3, 4). The light cells contained many vesicles of the smooth ER (Fig. 4). The dark cells had many free RNP particles within the cytoplasm (Figs. 3, 4). Golgi Zone The Golgi zone was found in different regions of the cell and was composed of the characteristic flattened sacs, vacuoles, and small vesicles. Occasionally melanosomes were seen in the Golgi vesicles (Fig. 6). Mitochondria The variation in size and distribution within the cells has already been noted. The mitochondria have the typical structure of an outer smooth membrane and highly infolded inner membrane (Fig. 7). Melanin Granules The melanin granules were very electron-dense and generally appeared oblong. The granules

3 ULTRASTRUCTURE OF PIGMENTED MELANOMA CELLS IN CONTINUOUS CULTURE 69 ti 4 FIG. 2. A cross-section of a dendritic process of a melanin-filled cell. Various stages of melanin granule formation are shown: (1) longitudinal and cross section of pre-melanosome, (2), (3), (4) longitudinal and cross section of melanosomes at varying degrees of melanization, (5) longitudinal and cross section of mature melanin granule. (Arrows with consecutive numbers show development of the melanin granules.) Note the membrane enclosing the melanin granule (arrows) (x 24,000).

4 70 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY '$ fn. sy r FIG. 3. A comparison of the cytoplasm of the light and dark cells. Note the high concentration of RNP particles in the dark cell. Rough endoplasmic reticulum (rer), melanin granules (mg), melanosomes (ms), are indicated (X 23,000). were enclosed by a membrane which wns tightly thin parallel rods which vnried in size. Crossbound to the grnnule (Fig. 8), or wns more loosely sections of the rods were frequently seen (Figs. fitting (Fig. 2). The internal structure of the 2, 8). Tn some granules the rods appeared to granule in longitudinal section appeared as exhibit n regulnr periodicity. Structures which

5 ULTRASTRUCTURE OF PIGMENTED MELANOMA CELLS IN CONTINUOUS CULTURE Vu4 I - FIG. 4. A section through a light and a dark cell showing the villus-like projections of the dark cell as compared to the smoother outline of the light cell. The light cell has an abundance of smooth endoplasmic reticulum (X 23,000). are probably the premelanosomes as named by Birbeck (7) were observed and they consisted of a membrane lined sac with parallel rods (Figs. 2, 8). These premelanosomes had very little electrondense substance within them. Various stages of melanin granule formation were observed from the premelanosomes to the mature melanin granules (Figs. 2, 8).

6 72 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY FIG. 5. A section through the nucleus and cytoplasm showing the double membrane of the nuclear envelope and melaoosomes and melanin granules (X ). Unidentified Electron-Opaque Bodies These irregular-shaped, electron-opaque bodies were occasionally seen in some of the cells. Their identity at present is not known (Fig. 9). DIscussIoN The cells of mouse and human melanumas have been extensively studied with the electron microscope (8 17). In the biopsy material, a mixture of

7 ULTRASTRUCTIJRE OF PIGMENTED MELANOMA CELLS IN CONTINUOUS CULTURE 73 V. 4-)' as FIG. 6. Cytoplasm of a light cell with a Golgi complex having flattened cisternae, vacuoles and smaller vesicles. Note the vacuoles containing melanin near the Golgi area (arrows) (X 30,000). cell types has been described which included pigmented and non-pigmented melanoma cells, fibroblasts and macrophages. When these tissues were explanted in vitro with the tissue culture technic, the cells in the outgrowth, when examined with the electron microscope, were found to possess ultrastructures similar to those of the cells prepared directly from the original tumors.

8 74 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY. 'I. 4 hi FIG. 7. Cytoplasm of a dark cell with an abundance of mitochondria (X 24,000). The cell strain used in this study included a to be only morphological variations of the same pigmented and non-pigmented cell type as re- cell, the melanocyte, at different stages of vealed by light microscopy. Even though the development and physiological activity (21, 22). morphology of these cells varied from round, The small round cells probably represent the ovoid, stellate to polydcndritic, they are believed young cells which, as they develop further,

9 ULTRASTRUCTUEE OF PIGMENTED MELANOMA CELLS IN CONTINUOUS CULTURE 75 w*n a' c a:.. ras-aa a *'1 Ill-ps. FIG. 8. A section of melanin-containing cell showing the structure of the mature melanin granules. Pre-melanusomes and melanosomes are also seen (X 24,000). become ovoid, stellate and polydendritic. In the course of their development they also acquire the capacity of melanin production. Under electron microscopy three cell types are recognized, depending on their cytoplasmic ultrastructural variations. These cell types could also be interpreted as cells of the same type, but in different stages of development and functional activity. Mcrcer (23) discussed the classification of cells

10 76 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Fm. 9. A section through a cell containing irregular osmiophilic bodies (ob) and large vacuoles (v) (X 24,000). in terms of their protein synthesizing mechanisms. He distinguished: (1) growing and dividing cells which synthesize material principally to form new cellular structures, (2) secretory cells which produce protein, mucin, etc. for secretion and (3) retaining cells which synthesize proteins that remain with the cell, e.g., myoblasts, erythroblasts, etc. The secretory cells are char-

11 ULTRASTRUCTURE OF PIGMENTED MELANOMA CELLS IN CONTINUOUS CULTURE 77 acterized by a remarkable gathering of rough endoplasmic reticulum in the form of paired particle-covered membranes and well-developed Golgi. The retaining cells have a much simpler cytoplasmic pattern. The rough ER clusters are absent, the Golgi is poorly developed and the cytoplasm is tightly packed with free RNP particles. The embryonic cells are the precursors of all other cells and should be considered as a special type of retaining cell, since to grow and divide the cells must synthesize and retain the proteins required to form daughter cells. Their cytology has the retainer pattern, namely an active nucleus, little rough ER and many free RNP particles. Another feature of the embryonic cell is the evidence of poor intercellular adhesion shown by the frequent occurrence of wide intercellular gaps between the cell membranes and their convoluted surface with microvilli. Also, he studied the fine structure of the tumor cells and concluded that the tumor cell contains all the structural elements of normal cells, especially in the well differentiated tumor. In the more anaplastic tumors, signs of cytological disorganization which include loss of intercellular adhesion can be detected. The variations in the amounts of certain cytoplasmic organdies apparently depend more on the physiology and functional activity of the cells. The lack of certain organelles or the abundance of others does not necessarily mean malignancy. All dividing cells (embryonic, renewal tissue cell and tumor cell) share the same characteristics. Following the same line of thought as suggested by Mercer we have attempted to classify the cells in this melanoma cell line. The light cells which are characterized by the numerous vesicles and complex cndoplasmic reticulum and well developed Golgi apparatus appear to fit the description of a secretory cell. The dark cells which show an abundance of free RNP particles in the cytoplasm and poorly developed endoplasmic reticulum and Golgi apparatus probably belong to the retaining cell type. These cells are also observed to have more villous projections at the free surface of the plasma membrane, a feature possessed by the dividing or embryonic cells. The completely melanized cells, of course, represent the fully developed mclanocytes with accumulation of secretory particles. In electron microscopic examinations of cells of human, as well as mouse and hamster melanomas, Wellings and Siegel (16) observed "pale" and "dark" mclanocytes. The "dark" mclanocytes possessed cytoplasm of high density, apparently due to a greater concentration of RNP per unit area. In human melanomas both types were frequently encountered; in mouse and hamster melanomas, the "pale" mclanocytcs predominated. An abundance of RNP particles was present on the outer surface of the crgastoplasmic sacs as well as scattered through the intervening cytoplasmic matrix. The space inside the sacs frequently contained very fine particulate material of low density, measuring 50 to 100 A or less. Similar particles were present in abundance within the flattened sacs, microvesicles, and vacuoles of the Golgi apparatus. They advanced the hypothesis along with others (4, 9, 24), that Golgi apparatus is the principal site of melanin granule formation. In view of the similarities between melanin granule formation and the formation of the other types of visible particulate secretory products, for example zymogen (25) and casein (26), the authors suggested that melanocytes and their neoplastic counterparts be regarded as specialized secretory cells. The electron microscopical observations of melanocytcs of human hair by Birbeck (7) also suggest that melanin granule synthesis occurs in the Golgi zone of the cell. The fine structures of the premelanosomes, melanosomes and mature melanin granules in our cultured cells correspond closely to those described by other workers (7, 16, 17). The crystalline-like lattice structure described by Drochmans (5) in his permanganate-treated sections was not seen in our material. The difference may be due to the different staining methods used, since in his osmium tetroxide and PTA-stained section, only longitudinal striations were observed. A flat pouch-like structure has been described by Zelickson and Hartman (6) in the human cpidcrmal mclanocytes. Since detailed description of this structure is not available, it is not certain whether such structures arc present in the material we have examined. It is impossible to state with certainty which cell is the progenitor of the melanin-filled cell or the mature melanocyte. On the basis of ultrastructural evidence both the light and the dark cell possess cytoplasmic organclles which arc important for protein synthesis. However, the light cell has the higher concentration of Golgi

12 78 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY and endoplasmic reticulum which have been demonstrated to be of primary importance in the formation of secretory material in the guinea pig pancreas (27, 28, 29, 30). Assuming that the light cell is more actively forming melanin granules and is eventually converted to a melaninfilled cell (mature melanocyte), the dark cell may be the less differentiated cell and is involved in division to form more dark and light cells. One may propose that the dark cell forms a light cell which fills with melanin granules to become the mature melanocyte. This material presents a good opportunity to investigate this question further. The evidence for the formation of melanin granules is considerably stronger. From the electronmicrographs of these cells it appears that the melanin granules begin as a vacuole with fine rods or fibers contained within the vacuole. The site that these vacuoles or premelanosomes first appear within the cell is not certain; but frequently they are found near definitive Golgi zones. The implication is that the premelanosomes have their origin within the Golgi complex as proposed by Birbeck (2, 4, 7, 23), Wellings and Siegel (10, 16), and Dalton and Felix (9). The vacuole fills with melanin by growth of the rod structures and eventually becomes a mature melanin granule. The intermediate stage is the melanosome. SUMMARY AND conclusions A pigment cell line established from B 16 mouse melanoma in tissue culture was studied with the electron microscope. Three cell types were identified. These cell types represent the various developmental stages of the pigment cell. The light cells probably are amelanotic melanocytes and the dark cells represent the active dividing cells, perhaps the melanoblasts. The melanin-containing cells are the mature, full-grown melanocytes. The endoplasmie reticulum and Golgi apparatus are believed to be the structures involved in melanin synthesis. Various stages of melanin granule formation, namely, premelanosomes, melanosomes, and mature melanin granules, were observed and described. The similarity between the ultrastruetures of the cultured mouse melanoma cells and those of the mouse and human melanoma cells and those of the human hair bulb melanoeytes is discussed. The cultured cells appear to fit the description and the properties of the melanoeytes and offer a relatively homogeneous cell population for the study of the fine structures of the pigment cells and melanogenesis in vitro. REFERENCES 1. CLARK, W. H., Ja. AND HIBBS, R. G.: Electron microscope studies of the human epidermis. The clear cell of Masson (deadritic cell or melanocyte.) J. Biophys. Bioehem. Cytol., 4: 679, BARNICOT, N. A. AND BIEBEcK, M. S. C.: The electron microscopy of human melanocytes and melanin granules. In: The Biology of Hair Growth. W. Montagaa and R. A. Ellis, Eds. pp New York, Academic Press CHARLES, A. AND INGRAM, J. T.: Electron microscope observation of the melanocyte of the human epidermis. J. Biophys. Biochem. Cytol., 6: 41, BIEBECK, M. S. C. AND BARNIeOT, N. A.: Electron microscopic studies on pigment formation in human hair follicles. In: Pigment Cell Biology. Myron Gordon, Ed. pp New York, Academic Press, DEOCHMANs, P.: Electron microscope studies of epidermal melanocytes and the fine structure of melanin granules. J. Biophys. Biochem. Cytol., 8: 165, ZELIcK50N, A. S. AND HANTMANN, J. F.: The fine structure of the melanocyte and melanin granule. J. Invest. Derm., 36: 23, BIRBEcK, M. S. C.: Electron microscopy of melanoeytes: The fine structure of hair bulb premelanosomes. Ann. N. Y. Acad. Sci., 100: 540, S. DALTON, A. J. AND FELIX, M. D.: Phase contrast and electron micrography of the Cloudman S-91 mouse melanoma. In: Pigment Cell Growth. Myron Gordon, Ed. pp New York, Academic Press, DALTON, A. J. AND FELIX, M. D.: The electron microscopy of normal aud malignant cells. Ann. N. Y. Aead. Sci., 63: 1117, WELLINGs, S. R. AND SIEGEL, B. V.: Role of Golgi apparatus in the formation of melanin granules in human malignant melanoma. J. T.Jltrastruet. Res., 3: 147, WELLINGs, S. R, BAEISHAK, R. AND SIEGEL, B. V.: Cytological studies of human malignant melanoma cultured in vitro. Cancer Res., 20: 347, WELLING5, S. R. AND SIEGEL, B. V.: Electron microscopy of human malignant melanoma. J. Nat. Cancer Inst., 24: 437, BAEI5HAK, R., WELLING5, S. R. AND SIEGEL, B. V.: Cytologic studies of cultured cells of the Cloudman S-91 mouse melanoma. Amer. J. Path., 38: 371, SEIJI, M. AND FITZPATRICK, T. B.: The melanosome: A distinctive subeellular particle of mammalian melanocytes and the site of melanogenesis. J. Invest. Derm., 36: 243, SEIJI, M., SHIMAD, K., BIRBECK, M. S. C.

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