Ultrastructural characteristics of a cell line derived from a melanotic neuroectodermal tumor of infancy
Lewis J. Ciaman\ David Stetson^ Barry Steinberg^ and Charies F. Shuler" Departments ot 'Periodontology. ^Zoology, Ohio State University, Cotumbus, ^Northeastern Ohio Universities College of Medicine, Rutstown, and 'Center for Craniofaciat Molecular Biology, University of Southern California, Los Angeles, California, USA.
Claman LJ. Stetson D. Steinberg B. Shuler CF: Ultrastructural characteristics of a cell line derived from a melanotic neuroectodermal tutnor of infancy. J Oral Pathol Med 1991: 20: 245-9. Thin section and freeze-fracture transmission electron microscopy were used to examine and identify the cytoplasmic and membrane structures in a cell line derived from a melanotic neuroectodermal tumor of infancy (MNTI). The cultured cells had a uniform appearance after 70 population doublings characterized by long dendritic processes and evidence of melanin production. The cytoplasm contained numerous melanosomes in various stages of developtnent. vesiculated rough endoplasmic reticulutn, microfilatnents and uncoated as well as coated vesicles. The membrane specializations included caveoli. coated pits, gap junctions, microfilaments, desmosome-like structures and latnellipodia. The ultrastructural appearance of the cultured MNTI cells was similar to features previously seen in electron micrographs of MNTI tutnor specimens. However, correlated freeze-fracture and thin section micrographs permitted further identification of structures previously described. The MNTI cell line represents one of the cell types of the tutnor and provides an opportunity for further study of the pathogenesis of this rare tumor.
The melanotic neuroectodennal tumor of infartcy is a rare lesion that occurs most often in the maxilla of children under the age of I yr (1-3). While very rare cases of malignant tumors have been reported, the lesions are generally considered to be benign with the possibility of local recurrences (1-7). The classic tumor has a biphasic histoiogic pattern with nests and cords of stnall basophilic cells surrounded by spindle shaped cells that tnay contain melanin (1-7). The tumor cells appear to be of neural crest origin but the actual tissue of origin has not been determined. Previously different techniques have been used in attempts to characterize the tumor cells. Electron microscopic studies of tumor tissue have shown that as many as four different types of cells can be identified within the parenchyma of a tumor (4-6). Cells frotn one tnalignant MNTI tumor have been maintained for a short period in tissue culture (4). These investigators were able to differentiate three types of cells in the primary cultures, one of which was pigmented.
We have described previously the clinical course of a melanotic neuroectodermal tumor of infancy (MNTI) which developed a recurrence requiring aggressive therapy (7). At the initial surgical procedure, portions of the tutnor were obtained and placed in tissue culture. The objective of the present study was to characterize the ultrastructure of a cell line which was derived from a MNTI tumor. Freeze-fracture transmission electron microscopy was used to define the morphology of the cell surface more precisely than is possible with thin section ultrastructural tnethods. Thin section electron microscopy was used to examine the cell-cell and cellsubstrate contacts.
iVIateriai and methods
A 10-wk-old white boy presented with a rapidly expanding 1.5 x 1.5 cm mass of the left anterior maxilla (For a complete description of the clinical case and a review of the clinical literature, refer to reference 7). The tumor was treated
Key words: melanotic neuroectodermal tumor of infancy: cell line: freeze-fracture transmission electron microscopy. Charles F. Shuler. Center for Craniofacial Molecular Biology, University of Southern California, 2250 Aleazar Street, CSA 103, Los Angeles, California 90033. USA Accepted for publication December 30, 1990.
by local excision and portions of the tumor were taken to the cell culture laboratory in sterile saline solution. The tumor exhibited the classic histoiogic features of MNTI (7). Cell cultures were initiated from pritnary explants of tissue obtained from the surgical specimen of the tumor. The tissue was minced to produce 1 x 1 tnm fragments. These pieces were allowed to adhere to the bottom of Primaria dishes (Falcon). The cultures were nourished with a mediutn that consisted of Dulbecco's Minimal Essential Medium (MEM) (Gibco) supplemented with 10% fetal bovine serum and antibiotics and tnaintained at 37X in 5% CO,. Subconfluent cultures were passaged by releasing the cells from the tissue culture dishes with 0.25% trypsin and 10 mM EDTA. The subpassaged cells were tnaintained in the same medium and environment. Cells were frozen at each subpassage and stored in liquid nitrogen to maintain stocks of cells of different passage nutnber. The pattern of growth of the tumor cells was monitored in two ways. Cell
246
CLAMAN et al.
counts were made at each subpassage to determine the rate of growth of the cells. Phase contrast microscopy was used to examine and photograph the morphology of the cultured cells. The cells used for ultrastructural examinations were monomorphic. nearly confluent and had been passaged between 5 and 7 times. The cultured cells, to be examined by thin-section electron microscopy, were fixed and processed in situ in the culture dish. The cells were fixed isotonically with gluteraldehyde (1.6% in 0.05 M phosphate buffer, and 0.05 M sucrose for 30 min), postfixed in osmium tetroxide (1%, 30 min), dehydrated through graded alcohols, stained en bloc with aqueous uranyl acetate (2%) and embedded in Epon 812. After embedding, the plastic culture dish was removed. The cells were sectioned parallel to the culture dish surface and the sections stained with uranyl acetate and lead citrate. The sections were examined with a Phillips 300 electron microscope. In addition to thin .sections, cultured MNTI cells were prepared for freeze fracture transmission electron microscopy. Use of the monolayer freeze-fracture technique permitted minimal manipulation of the cells prior to fracture (8, 9). Nearly confluent cultures of MNTI cells were subpassaged in culture dishes containing 4 mm glass disks (Balzer Intl.). These cultures were subpassaged at a 1:2 ratio and allowed to grow on the disks for 20 h. The cultured cells for freeze-fraeture were fixed (2.5% gluteraldehyde in 0.1 M Sorenson"s phosphate buffer. 15 min). washed in buffer and then cryoprotected (25% glycerol. 30 min). The disks were sandwiched to double height planchets (Balzer Int.) in order to favor a fracture through the monolayer. The specimens were frozen in liquid Nj slush, placed in a double replica device under liquid N2 and introduced into the vacuum chamber of a 400 T freeze fracture unit (Balzer Int.). The fracture of the specimens was accomplished at — 110°C without etching. A platinum-carbon mixture was evaporated at a 45 C angle to a thickness of 5 nm and a carbon support was applied at a 90 angle to a thickness of 15 nm. Organic material adhering to the replicas was digested with a dilute solution of sodium hypochlorite (12 hours). The replicas were washed, fioated onto 100 mesh formvar coated grids and viewed in a Philips 300 transmission electron microscope.
Results Cell growth and morphology
The cells rapidly adhered to the culture dishes and began to proliferate. The disaggregated tumor portions gave rise to a mixed population of cells in vitro. This population contained cells with several different morphologies (Fig. lA). The cells were not subjected to selective growth pressures but were allowed to replicate and subpassaged prior to confluency. With subsequent passages the cultured cells became monomorphic with a dendritic cell morphology (Fig. 1B). The cells had long processes which appeared to interact with other cells and had a granular cytoplasm. These cells stained positive for the presence of melanin by both Fontana-Masson and ferric ion uptake. When the cells approached confiueney they had a more oblong, less dendritic appearance. Subpassages of nearly confluent cells resulted in the reappearance of the elongated dendritic morphology. The specific morphologic patterns and rates of growth persisted through eight subcultures and approximately seventy population doublings. Thin section transmission eiectron microscopy
Several features of the cultured cells were revealed with this technique. Mela-
Fig. 2. Thin seetion electron micrographs of MNTI cells in culture. Cytoplasm contains many electron-dense pretrtelanosonies (PM) often associated with Golgi cotnplexcs (G). Rough endoplasmic reticulum (RER) is vesiculated. Bundles of microltlaments (arrowheads) course through cytoplasm particularly around nucleus (N). x 13,875.
nosomes at different stages of developtnent were observed (Fig. 2). Perinuclear Golgi complexes and vesiculated cisternae of rough endoplasmic reticulum
Fig. I. Phase contrast micrograph oi' MNTI cells in culture. A, early cultures had cells with several morphologies. B, after prolonged passage of cells one cell type which contained pigment and had long dendritic proeesses predominated. A: x 64. B: x 256.
MNTI cell ultrastructure iti vitro 247 were found frequently (Fig. 2). The endoplasmic reticulum displayed a spiral pattern of ribosomes in some of the sections (Fig. 3). Microfilaments. a portion ofthe cytoskeleton, were commonly observed (Figs. 2 & 3). The nucleus had a prominent nucleolus with rather small regions of heterochromatin (Fig. 4). Regions of close association of two cells had desmosome-like structures that differed from true desmosomes in the spacing of the apposing plasma membranes and the specialization of the intercellular materials (Fig. 4). Evidence for other intercellular junctions, associated with cell-cell communication and passage of small ions, was also found (Fig. 5). These junctions could not be positively identified in thin section micrographs, however freeze fracture micrographs confirmed their identity as gap junctions. Coated pits, which represent sites of receptor mediated endocytosis, were observed (Fig. 5) as were coated vesicles (Figs. 3, 5, 6). The migratory nature of the cultured cells was confirmed by the observation of lamellipodia (Fig. 6). Freeze-fracture transmission electron "licroscopy
Fig. 4. Thin section electron micrograph of 2 MNTI cells in culture showing membrane specializations and junctional complexes. Desmosome-like structures of each eell are adherent to amorphous extracellular material, external lamina (arrowhead). N. nucleus. X 15,700.
membrane. Cross fractures through the cytoplasm showed a typical variety of membrane-bound organelles including Golgi complexes and mitochondria with external and internal faces visible (10). Irregular membrane-limited structures occupied the same region of the cytoplasm as the melanosomes identified in thin sections (Fig. 7). The cell plasma membrane had several surface specializations. Smooth surfaced plasma membrane regions were observed at sites of attachment to the culture substrate (Fig. 8). Caveolae (uncoated pits) were commonly present in fractures through the plasma membrane (Figs. 8, 9). Upon separation of the caveoli from the plasma membrane they enter the cytoplasm and appear as uncoated vesicles in thin section, a feature previously described for the cultured MNTI cells. Large pits were also identified in fractures through the plasma membrane (Fig. 9). Gap junctions were present in variety of configurations (Fig. 9). One of these configurations was distinct with a circular junctional plaque surrounded by caveoli (Fig. 9). Discussion
Freeze-fracture replicas were obtained of both the cytoplasm and the plasma
In the present study the ultrastructural features of a melanin producing cell line
IS r , . •
Lewis J. Ciaman\ David Stetson^ Barry Steinberg^ and Charies F. Shuler" Departments ot 'Periodontology. ^Zoology, Ohio State University, Cotumbus, ^Northeastern Ohio Universities College of Medicine, Rutstown, and 'Center for Craniofaciat Molecular Biology, University of Southern California, Los Angeles, California, USA.
Claman LJ. Stetson D. Steinberg B. Shuler CF: Ultrastructural characteristics of a cell line derived from a melanotic neuroectodermal tutnor of infancy. J Oral Pathol Med 1991: 20: 245-9. Thin section and freeze-fracture transmission electron microscopy were used to examine and identify the cytoplasmic and membrane structures in a cell line derived from a melanotic neuroectodermal tumor of infancy (MNTI). The cultured cells had a uniform appearance after 70 population doublings characterized by long dendritic processes and evidence of melanin production. The cytoplasm contained numerous melanosomes in various stages of developtnent. vesiculated rough endoplasmic reticulutn, microfilatnents and uncoated as well as coated vesicles. The membrane specializations included caveoli. coated pits, gap junctions, microfilaments, desmosome-like structures and latnellipodia. The ultrastructural appearance of the cultured MNTI cells was similar to features previously seen in electron micrographs of MNTI tutnor specimens. However, correlated freeze-fracture and thin section micrographs permitted further identification of structures previously described. The MNTI cell line represents one of the cell types of the tutnor and provides an opportunity for further study of the pathogenesis of this rare tumor.
The melanotic neuroectodennal tumor of infartcy is a rare lesion that occurs most often in the maxilla of children under the age of I yr (1-3). While very rare cases of malignant tumors have been reported, the lesions are generally considered to be benign with the possibility of local recurrences (1-7). The classic tumor has a biphasic histoiogic pattern with nests and cords of stnall basophilic cells surrounded by spindle shaped cells that tnay contain melanin (1-7). The tumor cells appear to be of neural crest origin but the actual tissue of origin has not been determined. Previously different techniques have been used in attempts to characterize the tumor cells. Electron microscopic studies of tumor tissue have shown that as many as four different types of cells can be identified within the parenchyma of a tumor (4-6). Cells frotn one tnalignant MNTI tumor have been maintained for a short period in tissue culture (4). These investigators were able to differentiate three types of cells in the primary cultures, one of which was pigmented.
We have described previously the clinical course of a melanotic neuroectodermal tumor of infancy (MNTI) which developed a recurrence requiring aggressive therapy (7). At the initial surgical procedure, portions of the tutnor were obtained and placed in tissue culture. The objective of the present study was to characterize the ultrastructure of a cell line which was derived from a MNTI tumor. Freeze-fracture transmission electron microscopy was used to define the morphology of the cell surface more precisely than is possible with thin section ultrastructural tnethods. Thin section electron microscopy was used to examine the cell-cell and cellsubstrate contacts.
iVIateriai and methods
A 10-wk-old white boy presented with a rapidly expanding 1.5 x 1.5 cm mass of the left anterior maxilla (For a complete description of the clinical case and a review of the clinical literature, refer to reference 7). The tumor was treated
Key words: melanotic neuroectodermal tumor of infancy: cell line: freeze-fracture transmission electron microscopy. Charles F. Shuler. Center for Craniofacial Molecular Biology, University of Southern California, 2250 Aleazar Street, CSA 103, Los Angeles, California 90033. USA Accepted for publication December 30, 1990.
by local excision and portions of the tumor were taken to the cell culture laboratory in sterile saline solution. The tumor exhibited the classic histoiogic features of MNTI (7). Cell cultures were initiated from pritnary explants of tissue obtained from the surgical specimen of the tumor. The tissue was minced to produce 1 x 1 tnm fragments. These pieces were allowed to adhere to the bottom of Primaria dishes (Falcon). The cultures were nourished with a mediutn that consisted of Dulbecco's Minimal Essential Medium (MEM) (Gibco) supplemented with 10% fetal bovine serum and antibiotics and tnaintained at 37X in 5% CO,. Subconfluent cultures were passaged by releasing the cells from the tissue culture dishes with 0.25% trypsin and 10 mM EDTA. The subpassaged cells were tnaintained in the same medium and environment. Cells were frozen at each subpassage and stored in liquid nitrogen to maintain stocks of cells of different passage nutnber. The pattern of growth of the tumor cells was monitored in two ways. Cell
246
CLAMAN et al.
counts were made at each subpassage to determine the rate of growth of the cells. Phase contrast microscopy was used to examine and photograph the morphology of the cultured cells. The cells used for ultrastructural examinations were monomorphic. nearly confluent and had been passaged between 5 and 7 times. The cultured cells, to be examined by thin-section electron microscopy, were fixed and processed in situ in the culture dish. The cells were fixed isotonically with gluteraldehyde (1.6% in 0.05 M phosphate buffer, and 0.05 M sucrose for 30 min), postfixed in osmium tetroxide (1%, 30 min), dehydrated through graded alcohols, stained en bloc with aqueous uranyl acetate (2%) and embedded in Epon 812. After embedding, the plastic culture dish was removed. The cells were sectioned parallel to the culture dish surface and the sections stained with uranyl acetate and lead citrate. The sections were examined with a Phillips 300 electron microscope. In addition to thin .sections, cultured MNTI cells were prepared for freeze fracture transmission electron microscopy. Use of the monolayer freeze-fracture technique permitted minimal manipulation of the cells prior to fracture (8, 9). Nearly confluent cultures of MNTI cells were subpassaged in culture dishes containing 4 mm glass disks (Balzer Intl.). These cultures were subpassaged at a 1:2 ratio and allowed to grow on the disks for 20 h. The cultured cells for freeze-fraeture were fixed (2.5% gluteraldehyde in 0.1 M Sorenson"s phosphate buffer. 15 min). washed in buffer and then cryoprotected (25% glycerol. 30 min). The disks were sandwiched to double height planchets (Balzer Int.) in order to favor a fracture through the monolayer. The specimens were frozen in liquid Nj slush, placed in a double replica device under liquid N2 and introduced into the vacuum chamber of a 400 T freeze fracture unit (Balzer Int.). The fracture of the specimens was accomplished at — 110°C without etching. A platinum-carbon mixture was evaporated at a 45 C angle to a thickness of 5 nm and a carbon support was applied at a 90 angle to a thickness of 15 nm. Organic material adhering to the replicas was digested with a dilute solution of sodium hypochlorite (12 hours). The replicas were washed, fioated onto 100 mesh formvar coated grids and viewed in a Philips 300 transmission electron microscope.
Results Cell growth and morphology
The cells rapidly adhered to the culture dishes and began to proliferate. The disaggregated tumor portions gave rise to a mixed population of cells in vitro. This population contained cells with several different morphologies (Fig. lA). The cells were not subjected to selective growth pressures but were allowed to replicate and subpassaged prior to confluency. With subsequent passages the cultured cells became monomorphic with a dendritic cell morphology (Fig. 1B). The cells had long processes which appeared to interact with other cells and had a granular cytoplasm. These cells stained positive for the presence of melanin by both Fontana-Masson and ferric ion uptake. When the cells approached confiueney they had a more oblong, less dendritic appearance. Subpassages of nearly confluent cells resulted in the reappearance of the elongated dendritic morphology. The specific morphologic patterns and rates of growth persisted through eight subcultures and approximately seventy population doublings. Thin section transmission eiectron microscopy
Several features of the cultured cells were revealed with this technique. Mela-
Fig. 2. Thin seetion electron micrographs of MNTI cells in culture. Cytoplasm contains many electron-dense pretrtelanosonies (PM) often associated with Golgi cotnplexcs (G). Rough endoplasmic reticulum (RER) is vesiculated. Bundles of microltlaments (arrowheads) course through cytoplasm particularly around nucleus (N). x 13,875.
nosomes at different stages of developtnent were observed (Fig. 2). Perinuclear Golgi complexes and vesiculated cisternae of rough endoplasmic reticulum
Fig. I. Phase contrast micrograph oi' MNTI cells in culture. A, early cultures had cells with several morphologies. B, after prolonged passage of cells one cell type which contained pigment and had long dendritic proeesses predominated. A: x 64. B: x 256.
MNTI cell ultrastructure iti vitro 247 were found frequently (Fig. 2). The endoplasmic reticulum displayed a spiral pattern of ribosomes in some of the sections (Fig. 3). Microfilaments. a portion ofthe cytoskeleton, were commonly observed (Figs. 2 & 3). The nucleus had a prominent nucleolus with rather small regions of heterochromatin (Fig. 4). Regions of close association of two cells had desmosome-like structures that differed from true desmosomes in the spacing of the apposing plasma membranes and the specialization of the intercellular materials (Fig. 4). Evidence for other intercellular junctions, associated with cell-cell communication and passage of small ions, was also found (Fig. 5). These junctions could not be positively identified in thin section micrographs, however freeze fracture micrographs confirmed their identity as gap junctions. Coated pits, which represent sites of receptor mediated endocytosis, were observed (Fig. 5) as were coated vesicles (Figs. 3, 5, 6). The migratory nature of the cultured cells was confirmed by the observation of lamellipodia (Fig. 6). Freeze-fracture transmission electron "licroscopy
Fig. 4. Thin section electron micrograph of 2 MNTI cells in culture showing membrane specializations and junctional complexes. Desmosome-like structures of each eell are adherent to amorphous extracellular material, external lamina (arrowhead). N. nucleus. X 15,700.
membrane. Cross fractures through the cytoplasm showed a typical variety of membrane-bound organelles including Golgi complexes and mitochondria with external and internal faces visible (10). Irregular membrane-limited structures occupied the same region of the cytoplasm as the melanosomes identified in thin sections (Fig. 7). The cell plasma membrane had several surface specializations. Smooth surfaced plasma membrane regions were observed at sites of attachment to the culture substrate (Fig. 8). Caveolae (uncoated pits) were commonly present in fractures through the plasma membrane (Figs. 8, 9). Upon separation of the caveoli from the plasma membrane they enter the cytoplasm and appear as uncoated vesicles in thin section, a feature previously described for the cultured MNTI cells. Large pits were also identified in fractures through the plasma membrane (Fig. 9). Gap junctions were present in variety of configurations (Fig. 9). One of these configurations was distinct with a circular junctional plaque surrounded by caveoli (Fig. 9). Discussion
Freeze-fracture replicas were obtained of both the cytoplasm and the plasma
In the present study the ultrastructural features of a melanin producing cell line
IS r , . •
