Establishment of a human polyclonal oral epithelial cell line.

OBJECTIVE: To develop a human oral epithelial cell line to constitute a continuous source of cells readily available for human oral epithelial cell research. STUDY DESIGN: Oral epithelial cells from a 30-week gestational, stillborn male fetus were grown in serum-free medium and transfected by lipid-mediation with the shuttle vector plasmid, pZ189, containing the T-antigen coding region and replication origin from the SV40 virus. RESULTS: Resulting cultures produced foci of rapidly multiplying cells that failed to senesce, in contrast to controls. The transformed culture, designated GMSM-K, was polyclonal. The original culture possessed a normal human male karyotype, and the transformed line was largely hypotetraploid. Multiple clones, isolated from soft agar studies and low density plating, showed decreased doubling times. Electron microscopy and immunohistochemistry confirmed an epithelial phenotype. Cells did not generate tumors in nude mice. CONCLUSION: Few human epithelial cell lines are available to investigators and most are tumor-derived. The nontumor-derived GMSM-K line has value as a resource for human oral epithelial cell research.

Attenuation of the natural course of herpes simplex virus infection in human oral epithelial cell cultures by smokeless tobacco extracts suggests the possibility of a synergistic mechanism for carcinogenesis.

High prevalence of both tobacco use and latent herpes simplex virus type 1 suggests the opportunity for synergism between these agents as cocarcinogens. In this study, postprimary human oral epithelial cell cultures were infected with herpes simplex virus type 1 pretreated with 2% extracts of either loose leaf, moist, or dry snuffs. Cultures were subsequently periodically exposed to the tobacco. Parameters measured included percentage of cultures undergoing active virus production, onset and time course of cytopathic effects, and concentration of virus released into the media over time. Results showed inhibition of both herpes simplex virus-mediated cell lysis and viral replication by tobacco extracts. This is the first time that these phenomena have been demonstrated in normal human oral epithelial cells. The work described here provides evidence to support a hypothesis that herpes simplex virus type 1 and smokeless tobacco may act synergistically in oral carcinogenesis.

The effect on cell phenotype of the mutagenic peptide of herpes simplex virus type-1.

The transforming region of the genome of herpes simplex virus type-1 (HSV-1) encodes a peptide that raises the mutation frequency of cells. To find the effect of this peptide on cell phenotype, three types of cells were transfected with a shuttle vector plasmid that expressed the peptide. When immortalised rat fibroblasts were transfected they rapidly became anchorage-independent with high efficiency, but were not tumorigenic in nude mice. When monkey kidney cells were transfected, five clonal cell lines were isolated, of which one became anchorage-independent but was not tumorigenic in nude mice. When human oral keratinocytes were transfected they did not become immortalised. The peptide therefore induced some of the features of transformation in different cell types, but did not induce a malignant phenotype in any cell. This suggests that interaction with co-factors would be necessary for the peptide to contribute to the development of oral cancer.

Morphologic and growth effects of tobacco-associated chemical carcinogens and smokeless tobacco extracts on human oral epithelial cells in culture.

Epidemiologic studies show an increase in the use of smokeless tobacco but few in vitro studies have directly assessed the potential for smokeless tobacco-induced oral carcinogenesis. Oral keratinocytes were grown to 90% confluence from explants of human labial and gingival mucosa at 34 degrees C, 5% CO2 in defined media. Epithelial monolayers were subsequently subcultured and then treated for 1 hour with aqueous extracts of moist or leaf smokeless tobacco, or with 0.25 to 1.0 ng/ml of three common smokeless tobacco carcinogens: 4-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-1-butanone; N-nitrosonornicotine; and benzo(a)pyrene. Even though the controls and most treatment groups terminally differentiated, cells exposed to 4-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-1-butanone, N-nitrosonornicotine, and moist and dry extract continued to divide, maintained a differentiated phenotype for 8 1/2 to 10 weeks in culture, and displayed focal growth and morphologic changes suggestive of early stages in cell transformation.

Size and charge selective permeability defects induced in glomerular basement membrane by a polycation.

Polyethyleneimine (PEI) was given intravenously to rats followed by native ferritin or one of three cationic ferritins. After 15 min kidneys were fixed for electron microscopy. Controls (C) received vehicle without PEI, followed by the appropriate ferritin. PEI-induced permeability change was measured as the ratio (PEI/C) of counted ferritin particles within the glomerular basement membrane (GBM) in the corresponding PEI and control groups. The ratio PEI/C for each tracer was: NF (pI = 4.5-4.8)-107; CF (pI = 7.5-8.2)-3.3; CF (pI = 8.0-8.7)-1.7; and CF (pI = 8.7-9.0)-0.9. When ferritin localization in the subendothelial layer of the GBM was examined separately, PEI/C was increased for all ferritins including the most cationic species. After PEI, GFR and RBF decreased proportionately by half; thus, filtration fraction remained constant. Reduction of renal perfusion pressure to 40 mm Hg showed no alteration of ferritin permeation into the GBM. Thus, PEI effects on ferritin localization in the GBM could not be ascribed to renal hemodynamic perturbations. If the effect of PEI on GBM permeability were to be mediated exclusively by neutralization of the charge barrier, the index PEI/C should be increased for NF, but decreased for all CF. The results show a marked effect of PEI on the charge barrier (PEI/C greater than 100 for NF). But, PEI also enhanced total GBM permeation by CF (7.5-8.2) and CF (8.0-8.7), and increased subendothelial GBM permeation for CF (8.7-9.0). An inverse relationship of the effect of PEI to the cationic charge on CF was evident.(ABSTRACT TRUNCATED AT 250 WORDS)