Transfection of wild-type TP53 induces differentiation in human gingival carcinoma cells.

We investigated the effects of transfection of wild-type TP53 on the growth properties of a human gingival carcinoma cell line, KOSC-3, in which the TP53 gene is mutated at codon 248 and overexpressed. The wild-type TP53 expression plasmid, pCDM8-p53/neo and the control plasmid, pCDM8/neo, were each stably transfected into KOSC-3 cells by using the calcium phosphate method. The number of G418-resistant colonies from wild-type TP53-transfected cells was approximately half that from plasmid controls. Exogenous wild-type TP53 transcripts were identified in four of the 20 G418-resistant clones analysed by reverse transcription PCR. Although the growth rates of the wild-type TP53+ clones did not drastically change during log phase, their saturation density was significantly reduced. The wild-type TP53+ cells were morphologically flat and enlarged when cultured in vitro, and were less able to form colonies in soft agar. In nude mice, the wild-type TP53+ clones formed subcutaneous tumours with conspicuous keratinisation and notable cell death that was not manifested in the parental and plasmid control cells. These findings indicate that the wild-type TP53 gene, even when it coexists with a mutated form, may function as a growth suppressor and differentiation inducer under restricted conditions in gingival squamous cell carcinoma.

Establishment of human oral-cancer cell lines (KOSC-2 and -3) carrying p53 and c-myc abnormalities by geneticin treatment.

Two cultured cell lines derived from human squamous-cell carcinomas were established through xenografted tumors in nude mice by "Geneticin" treatment, which allows to eliminate contaminated mouse fibroblasts and obtain enriched tumor cells at the early stage of cultivation. Line KOSC-2 and KOSC-3 were each derived from a squamous-cell carcinoma of the oral floor and of the lower gingiva, respectively. Both lines grew in a cobblestone pattern, demonstrating their epithelial heritage. Giemsa-banding patterns by chromosome analysis confirmed that both lines are of human origin. Molecular analysis of cancer-related genes, including the Ha-ras, c-myc and p53 genes, was performed. KOSC-3 cells showed co-over-expression of p53 and c-myc mRNA, in addition to p53 point mutation at codon 248 with transition from CGG to TGG. However, loss of heterozygosity (LOH) on chromosome 17 was detected in both lines by Southern blotting. These cell lines provide a model for elucidating the mechanism involving p53 inactivation and c-myc-gene over-expression.

[Effective removal of the contaminating host fibroblasts for establishment of human-tumor cultured lines].

We report a useful method for the establishment of cell lines in vitro from human tumors. One of the obstacles to establishing pure human cancer cells in vitro is contaminating fibroblasts in cultures. This obstacle could be overcome by selective-growth control of fibroblasts treated with serum-free GIT medium and their selective elimination with antibiotic Geneticin (G418-sulfate). In this study, the process of establishing cultured cell lines from two oral cavity cancers is demonstrated using GIT medium and Geneticin. The comparative study of the growth of cells of the two oral cavity cancers and of two control normal fibroblast cell lines supported the selective growth inhibition and elimination of normal fibroblasts by the "G-G method".