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 hu man diploid fibroblasts (WHE-7) and skin fibroblasts from a patient wi th Li-Fraumeni syndrome (MDAH 087) and a mutant p53 allele were treate d 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 inducibili ty of unscheduled DNA synthesis. The same degree of cytotoxicity was o bserved 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 cel l cultures (AFB(1)-treated and controls) failed to escape from senesce nce, whereas three out of nine AFB(1)-treated cultures of MDAH 087 cel ls 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 day s [10 population doublings (PD)] after the first treatment. However, t he cells recovered with faster growth rates after similar to 100 addit ional 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)-treat ed cell lines exhibited altered morphologies, chromosome aberrations ( numerical and structural aberrations) and loss of the wild-type p53 al lele. 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 induc e immortalization. This model may be useful for studying mechanisms of chemically induced immortalization.