The present study was undertaken to determine the role of the tumor suppressor gene p53 in the transformation of the human breast epithelial cell (HBEC) line MCF-10F treated with chemical carcinogens in vitro. MCF-10F is a spontaneously immortalized diploid HBEC line, derived from a mortal cell strain designated MCF-10M. MCF-10F cells became neoplastically transformed by in vitro treatment with the chemical carcinogens 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (BP). DMBA and BP-treated cells gave rise to clones D3, D3-1, BP1 and BP1-E, respectively, all of which expressed colony formation in agar-methocel and high chemoinvasion index. BP1-E cells, derived from BP1, were tumorigenic in severe combined immunodeficient (SCID) mice. We designed this work utilizing this model in which isolated clones of cells express different stages of progression to neoplastic transformation for determining whether any specific phenotype was associated with alteration in the p53 tumor supressor gene. For this purpose, Southern blot, Northern blot, single-strand conformation polymorphism (SSCP) and DNA sequencing were used to detect mutations in the highly conserved exons 5-9 of the p53 gene. Whereas no changes were detected in any of the cells tested by Southern and Northern blot, SSCP analysis showed a conformational shift in exon 7 in the MCF-10F cell line, and in clones BP1, BP1-E, D3, and D3-1, derived from DMBA and BP treated cells, respectively. This shift was absent in MCF-10M cells, the mortal cells from which the MCF-10F immortal cells were derived, and in the placental DNA used as control. Sequence analysis using asymmetric PCR-amplified products of exon 7 and an antisense primer revealed an insertional mutation of thymine at codon 254 in MCF-10F cells and in transformed cells, but not in MCF-10M. These data indicate that the emergence of the immortalized phenotype was associated with a mutation of p53. DMBA- or BP-treatment did not induce additional changes in the p53 gene. The fact that the precursor of the immortalized MCF-10F did not present changes in p53, may indicate that the alteration of this tumor suppressor gene could be associated with the process of cell immortalization; this, in turn, might facilitate the neoplastic transformation of the cells by chemical carcinogens.

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