Neoplastic transformation of primary tracheal epithelial cell cultures

• Published in: Carcinogenesis (New York) 1983, Vol.4 (4), p.369-374
• Main Authors: Pai, S.Balakrishna, Steele, Vernon E., Nettesheim, Paul
• Format: Article
• Language: English
• Subjects: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Epithelium - drug effects; Methylnitronitrosoguanidine - toxicity; Neoplasms, Experimental - pathology; Rats; Rats, Inbred F344; Trachea - drug effects; Trachea - pathology; Tracheal Neoplasms - chemically induced; Tracheal Neoplasms - pathology
• ISSN: 0143-3334; 1460-2180
• DOI: 10.1093/carcin/4.4.369

Primary cultures of rat tracheal epithelial cells were treated with the chemical carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) to quantitatively study the early events during neoplastic transformation. Epithelial cells were dissociated from tracheas of specific-pathogen-free Fischer-344 rats and were plated on collagen-coated tissue culture dishes. To determine cytotoxicity, cells were exposed on day 1 to various concentrations of MNNG for 3 h and colony forming efficiency (CFE) was determined on day 7. MNNG at a concentration of 0.1 μg/ml did not decrease CFE as compared to the control cultures, whereas 1 μg/ml reduced the CEF by 75%. For transformation studies, primary cell cultures received single exposures to MNNG (0.1–0.6 μg/ml) or multiple exposures to 0.1 μg/ml of MNNG for 3 h between days 1 and 17. In carcinogen-exposed cultures, morphologically altered foci appeared on day 18, recognizable by high cell density. Transformation frequencies between 1 and 8% were observed depending on MNNG concentration. Cultures containing altered foci continued to grow during the third and fourth week when control cultures had ceased to proliferate and exfoliated from the dish. Over 40% of the cultures which received multiple exposures to MNNG acquired cell line status and could be subcultured ≥20 times. None of the 30 control cultures became cell lines. Seventy per cent of MNNG-exposed cell lines showed the anchorage independent growth phenotype at passage 20 as judged by growth in agarose. Four of 10 cultures exposed either 6 or 8 times to MNNG formed invasive squamous cell carcinomas at passage 20 upon inoculation into nude mice. Based on these and previous studies, we feel that unrestricted cell replication is an early key event in carcinogen-exposed epithelial cell populations, preceding neoplastic transformation.