AFLATOXIN B-1-INDUCED IMMORTALIZATION OF CULTURED SKIN FIBROBLASTS FROM A PATIENT WITH LI-FRAUMENI SYNDROME

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 B-1 (AFB(1)). Exogenous metabolic activation of AFB(1) 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 AFB(1) at 0.1, 0.3 or 1 mu g/ml for 2 h with a 2% PMS mixture. All WHE-7 cell cultures (AFB(1)-treated and controls) failed to escape from senescence, whereas three out of nine AFB(1)-treated cultures of MDAH 087 cells escaped senescence, MDAH 087 cells treated with 0.1 mu g/ mi of AFB(1) two or three times initially decreased in growth similar to 40 days [10 population doublings (PD)] after the first treatment. However, the cells recovered with faster growth rates after similar to 100 additional days and grew continuously. Both cultures were immortal, defined as continuous growth for over 300 PD. Cells treated once with 0.3 mu g/ml of AFB(1) also escaped senescence, although they had about a 230 day time lag before restoration of cell growth. The three AFB(1)-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 AFB(1) 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.