Fig. 1.

Dose- and exposure-dependent induction of carcinogenesis by PhIP. MCF10A cells were repeatedly exposed to dimethyl sulfoxide (0) or 1, 10 and 100 pmol/l (pM); 10 nmol/l (nM) and 1 μmol/l (μM) PhIP for 5, 10 and 20 cycles. (A) To determine acquisition of the cancer-associated property of reduced dependence on growth factors (RDGF), cells were maintained in low-mitogen medium for 10 days. (B) To determine acquisition of the cancer-associated property of anchorage-independent growth (AIG), cells were seeded in soft agar for 14 days. Cell colonies (≥0.5 mm diameter) grown in low-mitogen medium and soft agar were counted microscopically. Columns, mean of triplicates; bars, standard deviation. The Student’s t test was used to analyze statistical significance, indicated by *P < 0.05, **P < 0.01 and ***P < 0.001. (C) Lysates from cells exposed to PhIP for 10 cycles were prepared and analyzed by western immunoblotting using specific antibodies to detect levels of H-Ras, phosphorylated Raf-1 (p-Raf-1), Raf-1, p-Mek1/2, Mek1/2, p-Erk1/2, Erk1/2 and Nox-1, with β-actin as a control. All results are representative of three independent experiments.