Fig. 2.

Alumina nanoparticle-induced cell proliferation and transformation. Cells were left untreated or treated with alumina nanoparticles (diameter < 20 nm; dose calculated for uniform application over cell surface) or TPA (10 ng/ml; positive control) and a growth curve was established by daily counting of cell numbers. Alumina- and TPA-treated JB6 cells demonstrated increase in growth rate, compared with controls *P < 0.006; **P < 0.001 (A). Western blot analysis indicated significantly increased PCNA levels in alumina- and TPA-treated cells, compared with controls, at 72 h (P < 0.001) and 120 h (P < 0.006) (B). Mouse anti-actin monoclonal antibody was used as an internal loading control (B). Quantitative analysis of PCNA expression was performed. Results were averaged from three sets of independent experiments (C). Cell viability of JB6 cells exposed to alumina in vitro was determined using the 3-(4,5-dimethythiazol-2yl)-2,5-diphenyl tetrazolium bromide assay, a colorimetric measure of metabolic activity, which serves as an indicator of cell viability. Cells treated with alumina demonstrated an increase in cell viability (*P < 0.001; n = 6), compared with controls (D). Phase contrast microscopy images of transformed colonies of JB6 cells seeded in soft agar, untreated or treated with nanoparticles of alumina or 10 ng/ml TPA (positive control) (E). The number of transformed colonies was counted after 14 days. The images shown were taken at ×10 magnification. All colonies found in alumina- or TPA-treated cells contain an average of >50 cells, as determined by dissociation of the smallest colony in the alumina-treated cells with trypsin and counting with a hemocytometer. Quantitative analysis showed that alumina-treated cells demonstrated a significant increase in transformed colonies compared with controls (alumina: P < 0.05; positive control TPA: P < 0.001) (F).