Benzoapyrene-induced CYP1A1 expression in MCF-7 cells is S-phase dependent.

Abstract

Xenobiotic biotransformation is often facilitated by oxidative metabolism via the cytochrome P450 (CYP) mixed function oxidase system. The aromatic hydrocarbon receptor (AhR) is a ligand-activated transcription factor that may bind benzo[a]pyrene (B[a]P). Unbound AhR resides in the cytosol as a multiprotein complex with two heat shock protein 90 molecules and an immunophilin-like molecule. When bound, the ligand-receptor complex translocates into the nucleus where the AhR dissociates from the complex and binds to a nuclear protein Arnt (AhR nuclear translocator). The resulting heterodimer binds to dioxin responsive elements and elevates the transcription of various genes, including CYP1A1, CYP1A2 and CYP1B1. In order to examine the role of cell-cycle phase on CYP up-regulation, MCF-7 cells in a confluent state (G1-phase concentrated), in an exponential growth-phase (S-phase concentrated), or following 18-h exposure of an exponentially-growing culture to 0.4 μg/ml nocodozole (G2/M-phase concentrated) were exposed to 24-h B[a]P (0.5 μM) treatment. The quantitative gene expression of CDKN1A (P21WAF1/CIP1), BCL-2, BAX, CYP1A1, CYP1A2, and CYP1B1 was determined. In G1-phase concentrated and/or G2/M-phase concentrated cultures, B[a]P-induced up-regulation of CYP1A1 was markedly suppressed (maximum ≈30-fold elevations compared to control calibrator) following 24-h exposures compared to ≈150-fold elevations observed in S-phase concentrated cultures. Although not as marked, a similar profile of differential gene expression modulated by cell-cycle phase was observed with CYP1A2 and CYP1B1. Consistent with this finding, S-phase concentrated cultures exhibited up-regulated P21WAF1/CIP1 (≈20-fold), down-regulated BCL-2 (≈5-fold), and up-regulated BAX (≈2-fold); suggesting a marked reduction in B[a]P-damage induction, these effects were not observed in G1-phase concentrated and/or G2/M-phase concentrated cultures. AhR influences G1-phase progression through interaction with retinoblastoma protein and up-regulated AhR-dependent activation of CYP1A1 appears to be cell-cycle phase dependent. Interestingly, exposure to relatively non-cytotoxic B[a]P concentrations results in cells accumulating in S-phase suggesting that evasion of G1-phase arrest is a factor to the transforming activity of DNA-adduct-forming carcinogens. Although hormone-induced effects mediated via the oestrogen receptors ERα and ERβ (e.g. AhR-ER crosstalk) undoubtedly play an important role in the proliferative response, such observations that CYP1A1 may play a pivotal role in G1- to S-phase transition might suggest that in combination with characteristic genetic toxicological effects, exogenous ligands of the AhR, through disrupted cell cycle kinetics, could drive the clonal expansion of an initiated phenotype.