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Inhibition of benzo(a)pyrene-induced cell cycle progression by all-trans retinoic acid partly through cyclin D1/E2F-1 pathway in human embryo lung fibroblasts

Benzo(a)pyrene [B(a)P] is a potent environmental carcinogen, which induces cell cycle changes. All‐trans retinoic acid (ATRA) is a promising agent in prevention and treatment of human cancers. In the present study, we investigated the inhibition of B(a)P‐induced cell cycle progression by ATRA in human embryo lung fibroblast (HELF). Our results showed that after treatment with B(a)P, the expression of cyclin D1 and E2F‐1 were both increased significantly in HELF. There were almost no changes of CDK4 and E2F‐4 expression by treatment with B(a)P. As expected, pretreatment with ATRA could efficiently decrease B(a)P‐induced overexpression of cyclin D1 and E2F‐1. In a further study, we stably transfected antisense cyclin D1 and antisense CDK4 plasmid into HELF. The inhibition of cyclin D1 expression and the inhibition of CDK4 expression significantly impaired the B(a)P‐induced overexpression of E2F‐1 respectively. Pretreatment with ATRA, cells expressing antisense cyclinD1 or antisense CDK4 showed a lesser decrease of B(a)P‐induced overexpression of E2F‐1 compared with similarly treated HELF. Furthermore, flow cytometry analysis showed that B(a)P promoted cell cycle progression from G1 phase to S phase, while pretreatment with ATRA could inhibit B(a)P‐induced cell cycle progression by an accumulation of cells in the G1 phase. It was suggested that ATRA could block B(a)P‐induced cell cycle promotion partly through the cyclin D1/E2F‐1 pathway in HELF.

Downregulation of cyclin D1-CDK4 protein in human embryonic lung fibroblasts (HELF) induced by silica is mediated through the ERK and JNK pathway.

Silica is a factor in the induction of acute injury and chronic pulmonary fibrosis. In 1996, silica was also listed as a human carcinogen by the International Agency for Research on Cancer (IARC). However, the molecular mechanisms involved in its pathologic effects are not well understood. We found that exposure of human embryonic lung fibroblasts (HELF) to crystalline silica for 2 h decreased cyclin D1 and cyclin‐dependent kinase 4 (CDK4) expression levels. Extracellular signal‐regulated protein kinase (ERKs), c‐Jun NH2‐terminal amino kinase (JNKs), and p38 kinase, as well as their downstream transcription factor, AP‐1, had different effects on the regulation of expression levels of cyclin D1 and CDK4 alterations induced by silica.

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]P. METHODS Human embryo lung fibroblasts (HELFs) were treated with 2 micromol/L or 100 micromol/L B[a]P which were provided with some characteristics of transformed cells (T-HELFs). Cyclin D1, 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 (G1) phase cells decreased. CDK4 and E2F-4 expression did not change significantly. In 2 micromol/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 micromol/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. CONCLUSIONS Cyclin D1/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 micromol/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 micromol/L B[a]P, both cyclin D1 and CDK4 negatively regulate the E2F-4 expression.