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Neoplastic Transformation of Human Lung Fibroblast MRC-5 SV2 Cells Induced by Benzo[a]pyrene and Confluence Culture¹

Molecular Toxicology, Faculty of Medicine, Imperial College of Science, Technology and Medicine, London, SW7 2AZ, United Kingdom

Benzo[a]pyrene (BaP) is potent rodent carcinogen and a reputed human carcinogen. Although much is known about its metabolic activation leading to DNA damage, the mechanisms of its actions are not as well understood at a cellular level. In addressing this, we have established an in vitro model that follows the progression toward neoplastic transformation induced by BaP. The model uses immortal nontumorigenic human lung fibroblast MRC-5 SV2 cells as effectors, cocultured with a metabolically competent human lymphoblastoid line h1A1v2 (activator cells). Treatment of the coculture with BaP for 48 h induced a dose-dependent decrease in cloning efficiency of the MRC-5 SV2 cells; nevertheless, cultures continued to progress to confluence. At prolonged confluence culture (day 11), an elevation in the proportion of G₂-M phase cells was detected by flow cytometry. By day 15, the G₂-M phase peak disappeared, accordant with the appearance of a population with DNA content greater than the cells in G₂-M phase. These changes in DNA ploidy were coincident with changes in morphology, specifically the appearance of enlarged and irregular-shaped nuclei. Confluence culture of BaP-treated MRC-5 SV2 cells for more than 2 weeks resulted in cell death; however, a few colonies survived the crisis to reach confluence again after an additional 10–14 days. The number of death-resistant colonies was proportional to the dose of BaP, with the majority of the cells exhibiting abnormal morphology. The degree of morphological change progressively increased with successive rounds of confluence. Cells that survived three rounds of confluence adopted a vastly different morphology, becoming polygonal, spindle, or other irregular-shaped, and acquired the ability to form large dense clumps that grew in an anchorage-independent manner. In parallel experiments, treatment with the vehicle alone (DMSO) resulted in substantially less death resistance and lower numbers of high-density clumps. Our studies demonstrate that a single pulse treatment of human MRC-5 SV2 cells with metabolically activated BaP increased DNA ploidy, induced resistance to confluence-initiated cell death and morphological change, and was accompanied by substantial changes in growth pattern under prolonged confluence culture. The multiple-step nature of this process is characteristic of the development of neoplastic disease, and prolonged confluence seemed to play a pivotal role in selecting for carcinogen-induced transformants.

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