M Azuma1, T Tamatani, Y Kasai, M Sato. 1. Second Department of Oral and Maxillofacial Surgery, Tokushima University School of Dentistry.
Abstract
BACKGROUND: Based on morphologic and functional differences mainly, 4 distinct types of cells are recognized in human salivary glands. For a better understanding of cellular proliferation and differentiation of human salivary glands as well as carcinogenesis of salivary gland neoplasms, we attempted to establish normal human salivary gland cells in an in vitro system. EXPERIMENTAL DESIGN: Primary cultured human salivary gland cells were transfected with origin-defective mutant DNA of SV40. After 2 to 3 weeks of transfection, slowly expanding colonies consisting of small compact cells emerged, whereas mock-transfected cells did not grow any more and eventually entered crisis, followed by cell death. RESULTS: Using limited dilution technique, we isolated 4 cell clones with distinct morphology from a single colony. Morphologic observation of cells cultured on plastic dishes precisely revealed the characteristics of constituent cells of salivary glands; i.e., three cell clones showing cuboidal (NS-SV-DC), spindle (NS-SV-MC), and flattened (NS-SV-SC) morphology were similar to duct, myoepithelial, and squamous cells, respectively. A remaining cell clone showing polygonal in shape with numerous secretory granules (NS-SV-AC) resembled acinar cells. Characterization of cell clones by the ultrastructural examination and the search for specific antigens showed the similarity of NS-SV-DC, NS-SV-MC, NS-SV-AC, and NS-SV-SC to duct, myoepithelial, acinar, and squamous cells, respectively. Integration and expression of SV40 DNA were confirmed by Southern blot and indirect immunofluorescence staining. Anchorage-independent growth in soft-agar and tumorigenicity in nude mice were not recognized in all cell clones. CONCLUSIONS: These results demonstrate that establishment of cell clones with duct-, myoepithelial-, acinar-, or squamous phenotype was accomplished in the in vitro system, and that based on the evaluation of colony-forming ability in soft-agar and tumorigenicity in nude mice, these cell clones are considered to be non-neoplastic.
BACKGROUND: Based on morphologic and functional differences mainly, 4 distinct types of cells are recognized in human salivary glands. For a better understanding of cellular proliferation and differentiation of human salivary glands as well as carcinogenesis of salivary gland neoplasms, we attempted to establish normal human salivary gland cells in an in vitro system. EXPERIMENTAL DESIGN: Primary cultured human salivary gland cells were transfected with origin-defective mutant DNA of SV40. After 2 to 3 weeks of transfection, slowly expanding colonies consisting of small compact cells emerged, whereas mock-transfected cells did not grow any more and eventually entered crisis, followed by cell death. RESULTS: Using limited dilution technique, we isolated 4 cell clones with distinct morphology from a single colony. Morphologic observation of cells cultured on plastic dishes precisely revealed the characteristics of constituent cells of salivary glands; i.e., three cell clones showing cuboidal (NS-SV-DC), spindle (NS-SV-MC), and flattened (NS-SV-SC) morphology were similar to duct, myoepithelial, and squamous cells, respectively. A remaining cell clone showing polygonal in shape with numerous secretory granules (NS-SV-AC) resembled acinar cells. Characterization of cell clones by the ultrastructural examination and the search for specific antigens showed the similarity of NS-SV-DC, NS-SV-MC, NS-SV-AC, and NS-SV-SC to duct, myoepithelial, acinar, and squamous cells, respectively. Integration and expression of SV40 DNA were confirmed by Southern blot and indirect immunofluorescence staining. Anchorage-independent growth in soft-agar and tumorigenicity in nude mice were not recognized in all cell clones. CONCLUSIONS: These results demonstrate that establishment of cell clones with duct-, myoepithelial-, acinar-, or squamous phenotype was accomplished in the in vitro system, and that based on the evaluation of colony-forming ability in soft-agar and tumorigenicity in nude mice, these cell clones are considered to be non-neoplastic.