Aflatoxin B₁-induced immortalization of cultured skin fibroblasts from a patient with Li-Fraumeni syndrome

Abstract

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 microgram/ml for 2 h 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 microgram/ml of AFB1 two or three times initially decreased in growth approximately 40 days [10 population doublings (PD)] after the first treatment. However, the cells recovered with faster growth rates after approximately 100 additional days and grew continuously. Both cultures were immortal, defined as continuous growth for over 300 PD. Cells treated once with 0.3 microgram/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.

Telomerase activity during spontaneous immortalization of Li-Fraumeni syndrome skin fibroblasts

“…In addition, it has been reported (Tsutsui et al, 1995) that enhancement of the immortalization frequency of 087 cells could be obtained by treatment of cells with a¯atoxin B1 (AFB1) and X-ray treatment, (Tsutsui et al, 1997). The 087 cell lines never developed detectable telomerase activity (Figure 1a) in eight of eight independent immortalization experiments including three immortalized by AFB1 (Tsutsui et al, 1995), one immortalized by X-ray irradiation (Tsutsui et al, 1997) and in four spontaneous immortalization events, (present study and Bischo et al, 1990). From that immortalization study cells were derived from 087+AFB1 and of these, cells capable of inde®nite proliferation were also found to be telomerase negative (Figure 1a).…”

Li-Fraumeni syndrome – a molecular and clinical review

“…As they are cultured through progressive population doublings, they show an increased number of chromosome abnormalities of all types Yin et al, 1992;Rogan et al, 1995), loss of the wild-type allele (Yin et al, 1992;Rogan et al, 1995), changes in morphology and, rarely, spontaneous immortalization Rogan et al, 1995) accompanied by telomere elongation (Rogan et al, 1995). Spontaneous immortalization has also been reported in breast epithelial cells from a LFS patient with a germline TP53 mutation (Shay et al, 1995), and even Li-Fraumeni cells that fail to immortalize spontaneously can be induced to do so by treatment with various agents that have no effect on normal cells (Shay et al, 1995;Tsutsui et al, 1995).…”

Chromosome instability is a predominant trait of fibroblasts from Li-Fraumeni families

“…Immortal derivatives of two of the fibroblast strains have been widely studied (Yin et al, 1992;Dulic et al, 1994;Ford and Hanawalt, 1995;Rong et al, 1995;Tsutsui et al, 1995). Cells from three of the families had normal sensitivity to high-dose-rate (HDR) ionizing radiation (Little et al, 1987), but mutation-carrying fibroblasts from another extensively studied family with a germline mutation in codon 245 (Srivastava et al, 1990) were resistant to HDR radiation (Bech-Hansen et al, 1981).…”