Ultrastructural Comparison of Two Human Malignant Melanoma Cell Lines1

[CANCER RESEARCH 30, 2782-2790, November 1970] Ultrastructural Comparison of Two Human Malignant Melanoma Cell Lines1

Gerd G. Maul and M. M. Romsdahl Department of Pathology, Temple University Health Sciences Center, Philadelphia, Pennsylvania 19140 fG. G. M.Â¡, and Department of Surgery, M. D. Anderson Hospital and Tumor Institute, University of Texas, Houston, Texas 77025 Â¡M.M. R.]

SUMMARY investigation. Line LeCa, obtained from a metastatic malig nant melanoma originating on the back of a 56-year-old male The ultrastructure of 2 established human melanoma cell patient, had been in continuous culture for 2 years at the Unes (LeCa and MeGo) and their clones was investigated. All time of this study. Line MeGo was derived from metastatic strains of line LeCa had premelanosomes with the typical disease in the groin, secondary to a malignant melanoma of arrangement of "helical filaments" and with cross-striated the foot. It has been cultured continuously for approxi melanosomes; only the amount of melanosomes produced mately 1.5 years. Both lines were maintained in modified varied in the different strains. McCoy's 5A medium supplemented with 20% fetal calf The strains in line MeGo varied widely in the appearance of serum. Cloning was performed on both malignant melanoma their premelanosomes. The helical arrangement of the matrix lines which resulted in the establishment and propagation of filaments was missing, and cross-striated melanosomes were strains that were individually unique in regard to morphol not seen. Morphologically, premelanosomes tend to resemble ogy, growth, pigment production, and tyrosinase activity. lysosomes in varying degrees in the different strains. Because The ceUs for study were prepared for electron microscopy all strains of both Unes are dihydroxyphenylalanine positive 48 hr after subculturing into flacon plastic tissue culture and show acid phosphatase activity in melanosomes, two flasks. Specimens were fixed for 1 hr at room temperature in possible explanations are considered: (a) hydrolytic enzymes 3% glutaraldehyde (phosphate buffered, pH 7.4) (26) and function in the melanosome to terminate melanogenesis; or postfixed for 1 hr with 1% Os04 (phosphate buffered, pH (Â¿>)the enzymes indicate hydrolysis of melanosomes as they 7.4). Before rapid dehydration in ethanol, the cells were accumulate in the melanoma cells. covered with 2% uranyl acetate in water for 1 hr (13). They were then flat-embedded (1) in Epon (14). This technique INTRODUCTION allowed selection of single cells with the light microscope. Several established human malignant melanoma cell lines Sections were cut on an LKB Ultratome III and stained with exhibited differences in morphology, amount of melanin uranyl acetate and lead citrate. Observations were made on a produced, enzyme activity, growth rate, and cytogenetics Hitachi HU-ll-C electron microscope operated at 75 kV. (24, 25). Because the ultrastructure of the melanosome Samples for the histochemical demonstration of enzyme varies considerably with different melanomas (2, 3) in vivo, activity were fixed in glutaraldehyde for 15 min. For the it was decided to investigate different melanoma cell lines at demonstration of tyrosinase activity, monolayers of fixed the ultrastructural level. Novikoff et al. (20) have shown that melanoma ceUs were covered with freshly prepared 5 mM the premelanosomes of mouse melanoma contain acid phos DOPA2 (Mann Research Laboratories Inc., New York, N. Y.) phatase activity, and they therefore considered this organelle in 0.1 M phosphate buffer, pH 6.8, for 4 hr at 36Â°(18, a modified lysosome. The ultrastructure of 2 cell lines will 20, 22). For the demonstration of acid phosphatase, cells be presented since they differed considerably from each were fixed in cacodylate-buffered glutaraldehyde (3%). The other with respect to melanosome morphology, and since method of Gomori (7) was adjusted for monolayers of cells they also seem to indicate that most "melanosomes" are by incubation for 10 min only. The substrate was omitted in lysosomes in melanomas (16). the controls. After incubation, the cells were osmicated and MATERIALSAND METHODS processed as described above. Two human malignant melanoma ceU lines (LeCa, MeGo) RESULTS utilized in this study have been described previously (24, Low-magnification survey micrographs of the Golgi region 25). Cloned strains of these ceU Unes were used in this and high-magnification micrographs of typical premelano-

1This research was supported by Grants CA 11654 and GM 15887 2The abbreviations used are: DOPA, dihydroxyphenylalanine; RER, from the N1H. It was conducted in part at the M. D. Anderson rough-surfaced endoplasmic reticulum; SER, smooth endoplasmic Hospital and Tumor Institute, Houston, Texas. reticulum; GERL, Golgi apparatus, endoplasmic reticulum, and lyso- Received March 6, 1970; accepted July 29, 1970. somes, according to the terminology of Novikoff (19).

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1970 American Association for Cancer Research. Ultrastructural Comparison of 2 Melanoma Cell Lines somes in that region in 2 cell lines (LeCa and MeGo) were are thin and long. The cytoplasm is dense, and many studied. Also, different strains of each cell line were investi polysomes are present at the cell periphery, although few are gated. No ultrastructural difference was found between the present in the area of the Golgi apparatus. SER is more cloned strains of malignant melanoma cell line LeCa that extensively developed than in strain 19-4. could not be attributed to culture conditions or plane of Strain 19-4 demonstrates no dense material in tubular SER section. There was, however, a difference in the number of connected with premelanosomes (15). In strain Amel-4, melanosomes present. This report will deal primarily with however, it is obvious that the tubular SER (diameter is the heavily pigmented strain 19-4. approximately 200 to 400 A) contains the same electron- Line LeCa, strain 19-4, corresponds to the lines reported in dense material as the melanosomes (Fig. 4). There is no long-term tissue culture cells (32). The individual cells are highly ordered underlying matrix, but the electron-dense markedly dendritic and contain all stages of melanosome material appears granular or clumped. Only in a few melano formation. Some cells also contain autophagosomes, which somes was there any suggestion of an ordered substructure are filled with melanosomes. Premelanosomes of all represen (Fig. 4, arrow). tative stages of development are concentrated around the The survey micrograph of another strain of line MeGo, Golgi-centriole region of the cell. Fig. 1 shows a relatively strain Amel-4P (Fig. 5), exhibits the familiar arrangement of high ratio of premelanosomes with advanced melanization. "premelanosomes" in the area of the Golgi apparatus. It Some premelanosomes were also found in more "remote" cannot be stated with much certainty that the dark granules areas of the cell. The Golgi apparatus is extensively devel are melanosomes since some or even the majority of them oped. Single dictyosomes consist of approximately 4 to 6 may be lysosomes. The Golgi apparatus is extensive as in the cisternae. Often one side of the dictyosome is fenestrated other clones, but the tubular SER is most extensive in this (Fig. 1, parallel arrows). This was observed in all prepara strain (Fig. 5, right lower corner), and there is high vari tions studied (17). Mitochondria were long, narrow, and ability between single cells. The mitochondria are thin and frequently branched. There were only a few polysomes long in the Golgi area but differ in shape within areas of the present in the Golgi area and very little RER. cell that contain large amounts of lipid droplets. "Empty" areas appearing in Fig. 1 (upper left) might be At higher magnification (Fig. 6), the laminated body is due to the leaching out of glycogen in our preparation structurally comparable with a lysosome. The dark bodies method. Glycogen was easily discernible in all strains when (db) contain clumped (dbÂ¡) or rod-like (db2) electron-dense stained with periodic acid-Schiff (24). The ultrastructure of material reminiscent of melanosomes. In several instances, the premelanosomes in line LeCa showed the classical ar typical ultrastructural features of a lysosome and an early rangement of helical fibers. Most stages of premelanosomes premelanosome within 1 membrane-bound structure were are present in Fig. 2; only the very early stages as described seen (Fig. 7). Melanosomes were also observed together with previously (15) are not present in this micrograph. externally ingested particles like mycoplasma. These findings Structures that seem to be helical fibers in Melanosome 1 indicate that a membrane-delimited body with the structural (ml) can be interpreted as folded sheets. The observation of inclusion of a melanosome may be a lysosome. The demon a light line (in m2), which runs in the center of the stration of tyrosinase activity and acid phosphatase activity melanized matrix, demonstrates an area of less melanization. was therefore used in the hope of distinguishing between the 2 This may be the unmelanized center of the helix inasmuch organelles, i.e., melanosome and lysosome. as it is assumed that several helices fuse into a sheet. This Tyrosinase activity was found in an anastomosing network assumption is further supported by the observation of many of tubular membranes associated with the Golgi apparatus, equally spaced light dots in cross-sectioned melanized strands coated vesicles, and melanosomes of strain 19-4 (Fig. 9). The (not shown in this micrograph). detailed account of the localization of tyrosinase activity in In comparison of line LeCa with line MeGo, one major normal melanocytes will be published elsewhere (G. G. Maul, difference is that the strains of line MeGo produce less On the Possible Function of Coated Vesicles in Melano- pigment as judged by the pale color of the cell pellet, which genesis of the Melanocytes in Regenerating Fowl Feathers, led to their designation as amelanotic strains (Amel-4, Amel- J. Cell Biol., in press) (see also Refs. 20, 22, and 28 for 4P). However, single cells observed by light microscopy were demonstration of tyrosinase activity at the electron micro seen to produce melanosomes, as evidenced by a denser scopic level). Acid phosphatase activity was found in the region in the area of the Golgi apparatus. This may indicate same organelles as well as in several cisternae of some of the the strain Amel-4 from line MeGo was not a pure clone but dictyosomes. Fig. 8 shows melanosomes of line LeCa, strain consisted of a population of genetically different cells or 19-4, with reaction product between the melanized matrix that only a few cells express the potential for melanosome and in Golgi-associated tubular membranes (compare with formation. Fig. 2). However, not all melanosomes contain the reaction In Fig. 3, a cell is shown from strain Amel-4, which when product of acid phosphatase activity (arrow). It was present viewed by light microscopy contained a dense Golgi region. in mature forms more often, but statistical evidence for this The arrangement of the melanosomes is similar to that of observation is not yet available. line LeCa except that they are more irregular and appear to The tyrosinase activity as judged by the DOPA reaction be of uniform density. The relationship of premelanosomes was found in both strains of line MeGo. It was present in to the tubular SER is the same as described from strain 19-4 the Golgi-associated structures as in Une LeCa but was of line LeCa (15). Mitochondria that surround the Golgi area difficult to ascertain in the melanosomes because of their

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inherent density and the lack of ordered substructures. Novikoff et al. (20) and Seiji and Kikuchi (27) reported the Reaction product of the acid phosphatase activity was presence of acid phosphatase activity in premelanosomes. present in most melanosomes of both line MeGo strains. These results are confirmed for human melanoma in vitro, The apparent average size of mitochondria is seen in Fig. 1. although this seems to be the case only in more mature However, mitochondria were very long and thin as based on premelanosomes (preliminary results). If this can be verified the reconstruction of serial sections. For study of the length statistically, a membranous connection of SER to the pre of mitochondria, cells with well-developed, long, thin den- melanosomes might be assumed to exist at a stage of drites were selected. These were aligned so that section could melanogenesis of individual melanosomes later than we had be made parallel to the knife edge. With this technique, it previously anticipated (15), that is, after melanogenesis has was possible to estimate the length of mitochondria since ended in an individual premelanosome. If there is no mem they were approximately in the same plane. The mitochon branous connection of GERL to the acid phosphatase con drion shown in Fig. 11 is 9.3 M long. Reconstruction from 3 taining more mature premelanosomes, as our results indicate, serial sections show it to be at least 11 Â¡ilong. Fig. 11 shows acid phosphatase may be channeled into the early premel typical mitochondira found in all cell lines. They were the anosome in an inactive form or by a mechanism different only type observed in the strain 19-4. In Amel P-4, however, from that proposed (8, 19). an unusual type were also present. These mitochondria are In line MeGo, we see an arrangement of dark bodies similar shown in Fig. 10. They were never observed near the Golgi to that in line LeCa, but the characteristic substructures of area but were most frequently noted on the opposite pole of premelanosomes are missing. We are justified in considering the nucleus and in an area of lipid droplet accumulation. them melanosomes only by our knowledge that line MeGo is These areas were also rich in RER, the latter coming in close a DOPA-positive human melanoma cell line, which we deter contact with lipid droplets. A dark area on the lipid droplet mined by the distribution and size of the dark bodies, by (arrow) indicates the attachment. In this area, markedly their electron density, and by their connection to SER. dilated RER, which was densely packed with a fibrous Many of these characteristics also apply to lysosomes. The material, was observed. dark granules of strain Amel P-4 appear morphologically even more characteristic of lysosomes. Also, most of them DISCUSSION contain acid phosphatase activity. In a recent study, Okun et al. (21) reported peroxidase- Many investigations have dealt with melanogenesis and the mediated synthesis of melanin from tyrosine or DOPA in origin of the melanosomes. The generally favored concept is melanocytes. They claim that mammalian peroxidase may that vesicles are pinched off from the Golgi apparatus and have roles in initiating melanin synthesis in vivo. As the are in turn enlarged by a process of fusion (6). In earlier different strains of line LeCa and MeGo have not yet been investigations, Novikoff (19) described the relationship investigated for peroxidase activity, we cannot exclude the between the various parts of the GERL. This investigation, possibility that peroxidase has reacted with DOPA; but, in as well as the one by Holtzman et al. (8), suggests a view of the report of Okun et al. (21) both enzymes would mechanism for a transport system of precursor of enzymes indicate melanin formation. The terminology of a membrane- to the membrane-bound organdÃes by direct transport bound organelle, which has the structural arrangement of a through tubular membranes. A different scheme has been melanin granule, may become rather confused if it is named proposed by Jamieson and Palade (10, 11). They show that according to the enzymes that it contains. It may be called a small, smooth-surfaced vesicles derived from RER or the peroxisome in the presence of peroxidase (21), a premelano Golgi apparatus fuse with condensing vacuoles, which in some in the presence of tyrosinase, and a modified lysosome turn form the zymogen granules in pancreatic cells. in the presence of acid phosphatase (16). No convincing mechanism has been described from an As melanosomes and lysosomes cannot be distinguished by ultrastructural viewpoint, which could explain the transfer of the presence or absence of acid phosphatase, morphological precursors from the Golgi complex into the premelanosomes criteria seemed to be an additional approach to differentia if such a route is assumed to exist. However, the first tion between these 2 organelles. It became clear during the possible transfer mechanism was suggested by Novikoff et al. course of this investigation that it is difficult to discriminate (20), who found both acid phosphatase and tyrosinase between the structures of concern in certain strains. Since all activities in the premelanosomes and also in tubular mem strains were DOPA positive and contained acid phosphatase, branous structures that belong to the GERL complex. It has we can only speculate. We may interpret the electron-dense been suggested, on morphological grounds, that enzyme granules in the micrographs of the line MeGo (Fig. 3) as systems may be channeled from the Golgi complex through premelanosomes that lack the characteristic helical matrix tubular membranous structures into premelanosomes (15). onto which tyrosinase could be oxidized and polymerized. The demonstration of membranous connections of early This may account for the presence of electron-opaque premelanosomes, tubular elements of the SER, and possibly material in very thin membranous tubules. A backflow may the dictyosomes were the basis for this hypothesis. exist because no polymerization occurs onto a preexisting Novikoff et al. (20) considered the melanosome a modified matrix structure. Single deletions of genetic information in lysosome. The structural evidence presented in this study melanosome formation may be responsible for the produc also suggests that there is a close relationship between the tion of incomplete melanosomes. In superficial, spreading melanosomes and lysosomes in melanoma cells in vitro. melanoma there is a definite formation of the helical matrix

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1970 American Association for Cancer Research. Ultrastructural Comparison of 2 Melanoma Cell Lines but no apparent melanization (3). This is also an indication lipid accumulation. No significance can be inferred from this of the interruption of the normal sequence of events in observation. Similar mitochondria are described in brown melanosome formation in melanoma. The appearance of a adipose tissue (29â€”31). Differences between in vitro cell typical premelanosome and a laminated body, typical for Unes may not necessarily reflect equal differences under in lysosomes within one limiting membrane, might be explained vivo conditions. The correlation of in vivo structures of the by the fusion of the two. This may lead eventually to the same tumor should be attempted to evaluate the relevance of formation of autophagosomes if it is a regulated process to studies on cultured malignant melanoma cell Unes. Such eliminate premelanosomes. It does not seem to be a studies are in progress. mechanism for the introduction of acid hydrolases into individual premelanosomes, as it was observed only on rare REFERENCES occasions. The presence of acid phosphatase in membrane-delineated 1. Brinkley, B. R., Murphy, P., and Richardson, L. C. Procedute for Embedding in Situ Selected Cells Cultured in Vitro. J. Cell Biol., bodies constitutes an intracellular digestive system (4, 5). It 35: 279-283, 1967. is suggestive of protein degradation or the potential thereof. 2. Clark, W. H., and Bretton, R. Four Kinds of Melanocytes Seen in The functioning of melanosomes in the coloration of verte Electron Micrographs of Primary Human Malignant Melanomas. brae (23) makes it unlikely that an organelle is built, partly Federation Proc., 29: 758, 1970. degraded, and then used. The possibility must be considered 3. Clark, W. H., and Bretton, R. Correlation of Melanosome Fine that, in melanoma cells, melanosomes are degraded as they Structure with Malignancy in Human Melanomas. Proceedings of accumulate and cannot be transferred to keratinocytes. the VII International Pigment Conference 1969, in press, 1970. Melanocytes may have the option of transferring melano 4. DeDuve, C. Lysosome Concept. In: A. DeRenck and M. Cameron somes or digesting them in case of overproduction or (eds.), CIBA Foundation Symposium on Lysosomes, pp. 1-31. reduced uptake by keratinocytes. This means that acid London: J. & A. Churchill, Ltd., 1963. phosphatase containing melanosomes must be regarded as 5. DeDuve, C. and Wattiaux, R. Functions of Lysosomes. Ann. Rev. lysosomes and not "modified lysosomes" (20), a term that Phyaol., 28: 435-492, 1966. 6. Fitzpatrick, T. B., Miyamoto, M., and Tshikawa. K. The Evolu seems to imply that a lysosome has been modified during tion of Concepts of Melanin Biology. Arch. Dermatol., 96: evolution or ontogenesis of the melanosome. A breakdown 305-323, 1967. of melanin granules within melanocytes had previously been 7. Gomori. G. Microscopic Histochemistry, pp. 137-219, Chicago: suggested (9, 12). The appearance of autophagic vacuoles in University of Chicago Press, 1952. melanoma cells filled with melanosomes seems to be another 8. Holtzman, E., Novikoff, A., and Villaverde, H. Lysosomes and mechanism with the same result. They may also contain acid GERL in Normal and Chromatolytic Neurons of the Rat hydrolases (20). An investigation of the presence or absence Ganglion Nodosum. J. Cell Biol., 33: 419-435, 1967. 9. Hu, F., Swedo, J., and Watson, J. Cytological Variations of B-16 of acid phosphatase in premelanosomes of normal melano Melanoma Cells, Advan. Biol. Skin, 8: 549-579, 1967. cytes could result in suggestive evidence for these special 10. Jamieson, J., and Palade, G. Intracellular Transport of Secretory circumstances in melanoma cells. Fitzpatrick et al. (6) did Proteins in the Pancreatic Exocrine Cell. HI. Dissociation of not find acid phosphatase activity in isolated melanosomes Intracellular Transport from Protein Synthesis. J. Cell Biol., 39: from melanocytes in vitro and from retinal pigment epithe 580-588, 1968. lium of 10-day-old chick embryos. This was confirmed by 11. Jamieson, J., and Palade, G. Intracellular Transport of Secretory electron microscopic histochemistry (27). These findings Proteins in the Pancreatic Exocrine Cell. IV. Metabolic Require support the hypothesis that, in melanoma cells, melanosomes ments. J. Cell Biol., 39: 589-603, 1968. containing acid phosphates are degraded. The lack of acid 12. Kawamura, T., Tkeda, S., Mori, S., and Obata, H. Electron phosphatase in the retinal pigment epithelium of the chick Microscopic Findings Compatible with Those of the Lysosome was interpreted as being due to the presence of immature (Autophagic Vacuole) Revealed in the Melanocytes in Cases of pigment granules, but fully melanized and DOPA-negative Conspicuous Pigment Blockade. Japan J. Dermatol., Ser. B, 76: 705-719, 1966. melanosomes were found in retinal pigment epithelium of 13. Kellenberger, E., Ryter, A., and Sechand, J. Electron Microscopic 6-day-old chick embryos (G. G. Maul, unpublished observa Studies of DNA-containing Plasma. II. Vegetative and Mature tion). If acid phosphatase were present in fully developed Phage DNA as Compared with Normal Bacterial Nucleoids in melanosomes of normal melanocytes, an alternative explana Different Physiological States. J. Biophys. Biochem. Cytol., 4: tion seems likely. Acid hydrolases may be introduced into 671-678, 1958. the membrane-bound melanosomes to stop enzyme activity 14. Luft, J. H. Improvements in Epoxy Resin Embedding Methods. necessary for melanosome formation. This may, then, be J. Biophys. Biochem. Cytol., 9: 409-414, 1961. part of a size-limiting control mechanism for melanosomes 15. Maul, G. G. Golgi-Melanosome Relationships in Human Melano about which we know absolutely nothing. mas i/i Vitro. J. Ultrastruct. Res., 26: 163-176, 1969. On the ultrastructural level, there were only two note 16. Maul, G. G. Melanosome Lysosome Relationship in Human worthy differences between the cell lines besides the differ Melanoma Cells in Vitro. Federation Proc., 29: 758, 1970. 17. Maul, G. G., and Brinkley, B. R. The Golgi Apparatus in Mitosis ences in premelanosome morphology, that is, the great of Human Melanoma Cell in Vitro. Cancer Res., 30: 2326-2345, abundance of free polysomes in line MeGo and the 1970. appearance of mitochondria that are more or less round. They 18. Mishima, Y. Electron Microscopic Cytochemistry of Melanosomes deviate strongly from the normal appearance, which is very and Mitochondria. J. Histochem. Cytochem., 12: 784-790, long and thin. These round mitochondria were found near 1964.

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19. Novikoff, A. GERL, Its Form and function in Neurons of Rat 26. Sabatini, D., Bensch, K., and Barrnett, R. Cytochemistry and Spinal Ganglia. Biol. Bull., 121: 358, 1964. Electron Microscopy. The Preservation of Cellular Ultrastructure 20. Novikoff, A., Albala, A., and Biempica, L. Ultrastructural and and Enzymatic Activity by Aldehyde Fixation. J. Cell Biol., 17: Cytochemical Observations on B-16 and Harding-Passey Mouse 19-58, 1963. Melanomas. The Origin of Premelanosomes and Compound 27. Seiji, M., and Kikuchi, A. Acid Phosphatase Activity in Melano Melanosomes. J. Histochem. Cytochem., 16: 299-319, 1968. somes. J. Invest. Dermatol., 52: 212-216, 1969. 21. Okun, M., Edelstein, L., Hamada, N., and Donnellan, B. Histo- 28. Stanka, P., Kinzel, V., and Mohr, U. Elektronenmikroskopische chemical Studies of Conversion of Tyrosine and DOPA to Untersuchung Ã¼berdie Pramelanosomenentstehung an Melanom- Melanin Mediated by Mammalian Peroxidase. Life Sci., 9: 491-505, 1970. zellen in Vitro. Virchows Arch. Abt. B. Zellpathologie, 2: 91-102, 1969. 22. Perotti, M. E. Cytochemical Techniques in Electron Microscopic Studies of Melanin Synthesis. Proceedings "del V Congresso 29. Suter, E. The Fine Structure of Brown Adipose Tissue. I. Italiano di Microscopia Elettronica Bologna," 1965, pp. 37-40. Cold-induced Changes in the Rat. J. Ultrastruct. Res., 26: 216-241, 1969. Bologna: Tipografia del Siminario d. Padoza, 1965. 23. Portmann, A. Die Vogelfeder als Morphologisches Problem. 30. Suter, E. The Fine Structure of Brown Adipose Tissue. II. Verhandl. Naturforsch. Ges. Basel, 74: 106-128, 1963. Perinatal Development in the Rat. Lab. Invest., 21: 246-258, 24. Romsdahl, M. Establishment, Characterization, and Biological 1969. Properties of Human Malignant Melanoma Cell Lines Grown in 31. Suter, E. The Fine Structure of Brown Adipose Tissue. III. The Vitro. Dissertation, University of Texas, 1968. Effect of Cold Exposure and Its Mediation in Newborn Rats. 25. Romsdahl, M., and Hsu, T. Establishment and Biologic Properties Lab. Invest., 21: 259-268, 1969. of Human Malignant Melanoma Cell Lines Grown in Vitro. Surg. 32. Toshima, S., Moore, G., and Sandberg, A. Ultrastructure of Forum, 18: 78-79, 1967. Human Melanoma in Cell Culture. Cancer, 21: 202-216, 1968.

Fig. 1. Survey micrograph of the Golgi area of Une LeCa, strain 19-4. Premelanosomes are most abundant in this region. Mitochondria (Mit) are long and narrow. The Golgi apparatus is extensive. Single dictyosomes with one side often fenestrated consist of a varying number (4 to 6) of cisternae. A tangential view (large arrow) as well as a cross-section (small arrows) is seen in the lower left. There are few free ribosomes and very little RER in the Golgi area. SER is present but not extensive. Cross-sections of SER appear as vesicles. "Empty" areas are considered to be regions where glycogen was leached out (upper left). X 27,000. Fig. 2. Detail micrograph of premelanosomes in line LeCa, strain 19-4. An area with premelanosomes in different stages of development is shown. They exhibit the typical helical arrangement of the matrix. Several helices (ml) are interconnected to form sheets. If these sheets are sectioned longitudinally, they show a light line in their center (m2). X 78,000. Fig. 3. Survey micrograph of the Golgi area of une MeGo, strain Amel-4. The Golgi apparatus is extensively developed. Premelanosomes are abundant. They appear different from those in strain 19-4. of Une LeCa (Fig. 2). No distinction can be made between premelanosomes and melanusomes. All have approximately the same density, and only a few have a substructure suggestive of an ordered matrix (arrow). There are many free ribosomes in the Golgi area but little RER. X 21,000. Fig. 4. Detail micrograph of the area outlined in Fig. 3 (line MeGo, strain Amel-4). The premelanosomes differ from those in line LeCa, strain 19-4. There is no helical matrix. Tubular membranous elements contain material similar to that in melanosomes. X 110,000. Fig. 5. Survey micrograph of the Golgi area of Une MeGo, strain 4-P. This micrograph is typical of the Golgi area of the cytocentrum. The mitochondria appear thin and long with dilated cisternae and dense matrix. The Golgi apparatus, as well as tubular SER (lower right), is extensive. Melanosomes are found primarily in this vicinity. There are few free ribosomes and minimal RER. X 17,000. Fig. 6. Detail micrograph of area outlined in Fig. 5. Premelanosomes cannot be distinguished clearly from lysosomes. The laminated body (Ib) is structurally comparable with a lysosome. The dark bodies may contain clumped (dbl) or rod-like (db^) electron-dense material reminiscent of melanosomes. X 78,000. Fig. 7 The presence of lysosome-like structures and single early premelanosomes was occasionally observed within one delimiting membrane. X 67,000. Fig. 8. Reaction product of the acid phosphatase activity was localized in tubular anastomosing membranes and in melanosomes, (line LeCa, strain 19-4). It is deposited between the melanized strands or sheets. Arrow, melanosome without reaction product (unstained section). X 25,000. Fig. 9. Reaction product of DOPA-incubated melanoma cells (line LeCa, strain 19-4) was localized in tubular anastomosing membranes, which were usually in close apposition to dictyosomes (d), and also in premelanosomes (pm). X 50,000. Fig. 10. Line MeGo, strain Amel-4, large irregular mitochondria are shown as well as the usual type (arrow pointing down). The large ones are situated predominantly in areas with abundant lipid droplets. RER is in contact with lipid droplets (small arrow) and is markedly dilated (large arrow). The dilated RER was filled with a dense, fibrous material. X 30,000. Fig. 11. Mitochondria sectioned obliquely. They were found in a dendrite of line LeCa, strain 19-4, cell sectioned parallel to its long axis. X 25,000.

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Gerd G. Maul and M. M. Romsdahl

Cancer Res 1970;30:2782-2790.

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