Isolation of Differentiated Squamous and Undifferentiated Spindle Carcinoma Cell Lines with Differing Metastatic Potential from a 4‐Nitroquinoline N‐Oxide‐induced Tongue Carcinoma in a F344 Rat

Abstract

One differentiated squamous cell carcinoma (SCC) cell line (RSC3‐E2) and two undifferentiated tumor cell lines (RSC3‐LM and RSC3‐E2R) with different metastatic potential were established from a 4‐nitroquinoline N‐oxide (4NQO)‐induced differentiated SCC in F344 rat tongue. The RSC3‐E2 subline was isolated from a parental cell line (RSC3‐P) by single cell cloning in vitro, whereas the RSC3‐LM subline was isolated from a lung metastatic focus after subcutaneous (s.c.) injection of RSC3‐P cells. The RSC3‐E2R cell line was isolated from a lung metastatic focus following s.c. injection of RSC3‐E2 cells after X‐irradiation in vitro. The RSC3‐E2 cell line is keratinpositive and grows as a keratinizing tumor in nude mice, whereas RSC3‐LM and RSC3‐E2R cells are keratin‐negative, vimentin‐positive and form undifferentiated tumors. When s.c. injected into nude mice, the RSC3‐E2 cell line proved to be non‐metastatic, while the RSC3‐LM cell line was metastatic by both hematogenous and lymphogenous routes, and the RSC3‐E2R cell line was metastatic only hematogenously. In vitro relative growth rates and in vitro invasion activity of these cell lines were in the order RSC3‐LM>RSC3‐E2R>RSC3‐E2. Chromosome analysis revealed two peaks with modal chromosome numbers of 83 and 78 for RSC3‐P cells and single peaks at 83, 78 and 56 for RSC3‐LM, RSC3‐E2 and RSC3‐E2R cell lines, respectively. Common structural abnormalities on chromosome 11 were shared by all cell lines. Mutation analysis of the p53 gene using a yeast functional assay demonstrated RSC3‐LM cell line to have a point mutation at codon 269, whereas RSC3‐E2 and RSC3‐E2R had double mutations at codons 106 and 170 on each allele. These results suggest that the two undifferentiated RSC3‐LM and RSC3‐E2R tumor cell lines with different metastatic potential were generated from differentiated SCC cells via different genetic pathways as a consequence of tumor progression in vivo and in vitro, respectively. These cell lines should provide a useful model for understanding mechanisms of hematogenous and lymphogenous metastasis, as well as tumor progression of oral SCCs.