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Aflatoxin B1-induced immortalization of cultured skin fibroblasts from a patient with Li-Fraumeni syndrome
Department of Pharmacology, The Nippon Dental University, School of Dentistry at Tokyo 1–9–20 Fujimi, Chiyoda-ku, Tokyo 102, Japan
1Laboratory of Molecular Carcinogenesis, Environmental Carcinogenesis Program, National Institute of Environmental Health Sciences, National Institutes of Health PO Box 12233, Research Triangle Park, NC 27709
2Department of Tumor Biology, University of Texas, M.D.Anderson Cancer Center Houston, TX 77030, USA
3To whom correspondence should be addressed
To examine the mechanisms of immortalization in human cells, normal human diploid fibroblasts (WHE-7) and skin fibroblasts from a patient with Li-Fraumeni syndrome (MDAH 087) and a mutant p53 allele were treated with aflatoxin B1 (AFB1). Exogenous metabolic activation of AFB1 with rat liver post-mitochondrial supernatant (PMS) was used and the optimal treatment conditions needed were determined by the inducibility of unscheduled DNA synthesis. The same degree of cytotoxicity was observed with MDAH 087 cells and normal WHE-7 cells treated with AFB1 at 0.1, 0.3 or 1 (µg/ml for 2h with a 2% PMS mixture. All WHE-7 cell cultures (AFB1-treated and controls) failed to escape from senescence, whereas three out of nine AFB1-treated cultures of MDAH 087 cells escaped senescence. MDAH 087 cells treated with 0.1 µg/ml of AFB1 two or three times initially decreased in growth {small tilde}40 days [10 population doublings (PD)] after the first treatment. However, the cells recovered with faster growth rates after {small tilde}100 additional days and grew continuously. Both cultures were immortal, defined as continuous growth for over 300 PD. Cells treated once with 03 µg/ml of AFB1 also escaped senescence, although they had about a 230 day time lag before restoration of cell growth. The three AFB1 -treated cell lines exhibited altered morphologies, chromosome aberrations (numerical and structural aberrations) and loss of the wild-type p53 allele. Although immortal, the cells were non-tumorigenic in nude mice. Spontaneous immortalization of untreated MDAH 087 was not observed in this study. The results indicate that AFB1 treatment of cells from a Li-Fraumeni patient, but not cells from normal individuals, can induce immortalization. This model may be useful for studying mechanisms of chemically induced immortalization.
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