POSTER NO: 92

Induction of neoplastic phenotype in MRC-5SV2 human lung fibroblast cells by benzo(a)pyrene is preceded by DNA ploidy changes

Huijun Zhu, Nigel J. Gooderham
Imperial College of Science, Technology and Medicine, Molecular Toxicology, Faculty of Medicine, SAF Building, Imperial College, South Kensington, London, SW7 2AZ, U.K.

Benzo(a)pyrene (BaP) is one of the procarcinogens belonging to a group of polycyclic aromatic hydrocarbons (PAHs), generated from cigarette smoke. The activation of BaP requires metabolism by cytochrome P450 enzymes (CYPs), and in particularly CYP1A1. The effects of BaP and its metabolites on spindle formation, mitosis and DNA ploidy have been documented in rodent cells. The aim of the current study is to investigate whether BaP exerts similar effects on human cells.

Unlike rodent cells, human cells are notoriously resistant to acquisition of neoplastic phenotype when cultured in vitro. In our studies we have used the permanent cell line MRC-5SV2, derived from MRC-5, a human foetal lung stain, by transfection with SV40. Thus the functions of p53 and retinoblastoma protein (Rb) are disrupted in these cells. Under subconfluent culture, MRC-5SV2 cells display normal cell cycle distribution. Treatment of the cells with BaP ( 5-25 uM) for 48 h in the presence of irradiated h1A1 v2 cells (metabolic competent cells that express human CYP1A1) induced a dose-dependent inhibition of cell growth, measured by cloning efficiency assay. After 15 days under confluent culture, the treated cells developed abnormal cell cycle distribution, showing DNA content from < 2N - > 4N, detected by DNA flow cytometry. The changes in DNA content were consistent with alterations in morphology, showing heterogeneous nuclear and cell size. Under repeated prolonged confluent culture (subculture every 3 weeks), surviving cells gradually acquired neoplastic phenotypes, displaying heterogeneous morphology, forming high dense foci and gaining increased ability to grow in soft agar. In contrast, the majority of cells treated with vehicle (DMSO) exhibited normal growth cycle with only a small number of cells with DNA content < 2N on day 15. Eventually all control cells died under repeated prolonged confluent culture. The effects of BaP on MRC-5SV2 cell growth and morphology were minimal when h1A1 V2 cells were omitted.

Our observations of DNA ploidy and morphology changes suggest that BaP or its metabolites may induce incomplete spindle formation in human MRC-5SV2 cells. The apparent genetic instability renders cells an increased potential to undergo neoplastic transformation, presumably as a consequence of compromised p53 and Rb function.