Vitamin C Inhibits Benzo[a]pyrene-Induced Cell Cycle Changes Partly via Cyclin D1/E2F Pathway in Human Embryo Lung Fibroblasts

Objective To study the molecular mechanism of the inhibitory effects of vitamin C on benzo[a]pyrene （B[a]P）-induced changes of cell cycle in human embryo lung fibroblast （HELF） cells. Methods The stable transfectants, HELF transfected with antisense cyclin D1 and antisense CDK4, were established. Cells were cultured and pretreated with vitamin C before stimulation with B[a]P for 24 h. The expression levels of cyclin DI, CDK4, E2FI, and E2F4 were determined by Western blot. Flow cytometric analysis was employed to detect the distributions of cell cycle. Results B[a]P significantly elevated the expression levels of cyclin D 1, E2F1, and E2F4 in HELF cells. Vitamin C decreased the expression levels of cyclin D 1, E2F1, and E2F4 in B [a]P-stimulated HELF cells. Dose-dependent relationships were not found between the different concentrations of vitamin C （10, 100, 500, 1000, and 5000 lamol/L） and the expression levels of cyclin D 1, E2F1, and E2F4 in HELF cells. The expression levels of cyclin D1, E2FI, and E2F4 in B[a]P-treated transfectants were lower than those in B[a]P-treated HELF cells. The expression levels of cyclin DI and E2F4 treated with vitamin C and antisense cyclin D1 were decreased compared with those treated with antisense cyclin DI alone. The effects of vitamin C combined with antisense CDK4 on the expression levels of cyclin DI and E2FI/E2F4 were similar to those of antisense CDK4 alone. B[a]P progressed HELF cells from GI to S phase. Both vitamin C and antisense cyclin DI suppressed the changes of cell cycle progressed by B[a]P. However, antisense CDK4 did not attenuate the above changes. Vitamin C combined with antisense CDK4 markedly suppressed B[a]P-induced changes of cell cycle as compared with antisense CDK4. But the inhibitory effects of vitamin C combined with antisense cyclin DI on B[a]P-induced changes of cell cycle were similar to those of vitamin C alone or antisense cyclin DI alone. Conclusions B[a]P progressed HELF cells from G1 to S phase via intracellular signaling pathway of cyclin D I/E2F. Vitamin C may modulate this signaling pathway to protect cells from injury caused by B[a]P.

Different Patterns of Cyclin D1/CDK4-E2F-1/4 Pathways in Human Embryo Lung Fibroblasts Treated by Benzo[a]pyrene at Different Doses

Objective To investigate the roles of the cyclin D1/CDK4 and E2F-1/4 pathways and compare their work patterns in cell cycle changes induced by different doses of B[a]E Methods Human embryo lung fibroblasts （HELFs） were treated with 2 μmol/L or 100 μmol/L B[a]P which were provided with some characteristics of transformed cells （T-HELFs）. Cyclin D l, CDK4 and E2F-1/4 expressions were determined by Western blotting. Flow cytometry was used to detect the distribution of cell cycle. Results After B[a]P treatment, the proportion of the first gap （G 1） phase cells decreased. CDK4 and E2F-4 expression did not change significantly. In 2 μmol/L treated cells, a marked overexpression of cyclin D1 and E2F-1 was observed. However, in T-HELFs overexpression was limited to cyclin D1 only, and no overexpression of E2F-1 was observed. The decreases of G1 phase in response to B[a]P treatment were blocked in antisense cyclin D1 and antisense CDK4 transfected HELFs （A-D1 and A-K4） and T-HELFs （T-A-D1 and T-A-K4）. After 2 μmol/L B[a]P treatment, overexpression of E2F-1 was attenuated in A-D1, and E2F-4 expression was decreased significantly in A-K4. In T-A-D1 and T-A-K4, E2F-4 expression was increased significantly, compared with T-HELFs. The E2F-1 expression remained unchanged in T-A-D1 and T-A-K4. Condusions Cyclin DI/CDK4-E2F-1/4 pathways work in different patterns in response to low dose and high dose B[a]P treatment. In HELFs treated with 2 μmol/L B[a]P, cyclin D1 positively regulates the E2F-1 expression while CDK4 negatively regulates the E2F-4 expression; however, in HELFs treated with 100 μmol/L B[a]P, both cyclin D1 and CDK4 negatively regulate the E2F-4 expression.