الگوهای مختلف مسیرهای Cyclin D1/CDK4-E2F-1/4 در سلول بنیادی انسانی، سلول های فیبروبلاست ریه تحت درمان با بنزو هیدروکاربن سفید با دوزهای مختلف

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 μmol/L or 100 μmol/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 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. 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 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.

Polycyclic aromatic hydrocarbon (PAH) is a ubiquitously distributed environmental pollutant formed by incomplete combustion of organic matters, such as cigarettes, fossil fuels, wood and urban waste. B[a]P is an important member of PAHs, and its carcinogenic and mutagenic effects have been well documented in animals and mammalian cell systems[1]. It is metabolized by cytochrome P450 enzymes to mutagenic derivative (BPDE) which can form DNA adducts. Most of previous studies focusedon this property. Studies have shown that B[a]P-induced murine skin tumors exhibit frequent and characteristic G to T mutation in the p53 gene[2] and B[a]P induces changes of p53 and related proteins in mouse skin[3]. B[a]P-treated C3H10T1/2 cells also exhibit significant DNA damages and stable covalent DNA adducts[4]. In mammalian cells, proliferation is controlled by a series of positive and negative regulatory factors. The transition from G1 phase to the DNA synthetic (S) phase of the cell cycle is controlled by the accumulation and assemblage of D-type (D1, D2, andD3) cyclins with their partners CDKs[5]. Overexpression of cyclin D is capable of accelerating the transition through the G1 phase of the cell cycle. Cyclin D1 can positively regulate the activities of CDK4. Cyclin D1 in complex with CDK4 phosphorylates the product of the retinoblastoma gene, the retinoblastoma protein (pRb), a well known tumor suppressor. The phosphorylated pRb releases the E2F family that plays an integral role in cell cycle progression by inducing the expression of gene required for S phase entry, including those involved in DNA synthesis, such as S phase regulatory factor cyclin E, cyclin A, and CDK2[6]. All E2F family members bind the same DNA sequence, but additional levels of regulation have been observed as follows: E2F-1, E2F-2, and E2F-3 associate with pRb, and E2F-pRb complexes are found primarily in G1; E2F-4 and E2F-5 bind to pRb, p107, and p130[7-8]. A numberof genotoxic agents perturb the G1 events which mediate cell cycle progression, thereby resulting in cell cycle arrest before S-phase entry[9-10]. As reported, B[a]P induces G1 phase arrest in a p53-independent manner due to DNA damage and inhibits growth factor-stimulated DNA synthesis[11]. BPDE causes S phase arrest in p53 deficient cells and the arrest is Chk1 mediated and caffeine sensitive[12]. However, the response of HME87 cells to damages induced by BPDE involves neither apoptosis nor a G1/S arrest at least for the first 24 hours after the treatment[13]. After being stimulated by serum, the cyclin D1 expression and the relative activity of E2F of Swiss 3T3 cells are unperturbed by B[a]P-induced DNA damage[14]. All the above studies have demonstrated that the pathways involved in cell cycle changes caused by genotoxic agents are dependent on cell types, doses and durations of stimuli. Little is known about the effects of cyclin D1/CDK4 and E2F-1/4 on B[a]P induced cell cycle changes. In this study, we have investigated the roles of the cyclin D1/CDK4 and E2F-1/4 pathways and compared their work patterns in cell cycle changes induced by different doses of B[a]P.

Representative histograms of DNA content were shown in Fig. 1. In B[a]P-treated HELFs, a decrease in the percentage of G1 phase cells was observed. Cell cycles of A-D1 and A-K4 cells were also examined, in which no marked changes were observed in comparison with HELFs. The fractions of A-D1 and A-K4 cells in S phase decreased after B[a]P treatment. The decreased percentages of A-D1 andA-K4 cells in G1 phase were significantly lower than those in HELFs. These results indicated that B[a]P was capable of promoting the cell cycle from G1 phase to S phase in the first post-treatment 24 h. And the inhibition of cyclin D1 and CDK4 could entirely reverse the B[a]P-induced cell cycle changes in HELFs.Cell cycle changes have been identified above. To understand the pathways which induced these changes, the effects of B[a]P on G1 phase cyclin/CDK and E2F-1/4 were evaluated in HELFs. Western blot analysis showed that relatively low levels of cyclin D1 and E2F-1 were present in extracts from serum starved HELFs. B[a]P induced a marked increase in cyclin D1 and E2F-1 expressions (Fig. 2a, lane 2). The transfection of antisense cyclin D1 reduced the overexpression of cyclin D1 (Fig. 2a, lane 3). The overexpression of E2F-1 elicited by B[a]P disappeared (Fig. 2a, lane 4). It indicated that cyclin D1 positively regulated the E2F-1 expression in B[a]P-treated HELFs. The CDK4 expression remained unchanged in B[a]P-treated HELFs (Fig. 2b, lane 2). The transfection of antisense CDK4 decreased the expression of CDK4 significantly and increased the expressions of cyclin D1 and E2F-1/4 (Fig. 2b, lane 3). There was a marked higher expression of CDK4 and lower expression of E2F-4 in B[a]P-treated A-K4 cells than that in A-K4 cells without B[a]P treatment (Fig. 2b, lane 4). The transfection of antisense CDK4 caused markedly higher expressions of cyclin D1 and E2F-1 induced by B[a]P (Fig. 2a, lane 6). No significant changes were seen in HELFs transfected with the vector of antisense cyclin D1 and antisense CDK4 plasmids (data not shown). These results suggested that CDK4 negatively regulated the expression of E2F-4 inThe changes of cell cycle in T-HELFs were investigated. In serum starved conditions, the majority of HELFs were in G1 phase (Table 1). The S and G2 phase cell population increased significantlyAfter B[a]P treatment, the cells died increasingly. Four weeks later, cells treated with 200 μmol/L B[a]P were all dead. A few cells treated with 100 μmol/L B[a]P survived. It indicated that cultures induced the accumulation of metabolically related damages, which led to cells death. Surviving cells treated with 100 μmol/L B[a]P grew rapidly. As shown in Fig. 3a, in 6 weeks after B[a]P treatment, cells proliferated rapidly and exhibited a random orientation and crisis-crossed under serum starved conditions. Cells with DMSO proliferated slowly, and were spindle-shaped (Fig. 3b). B[a]P treated cells showed heterogeneous morphological changes. They were generally large and most strikingly contained large or multiple nuclei (Fig. 3a). After 12 weeks’ culture, they became smaller and more spherical than HELFs (Fig. 3c). These results implied that B[a]P induced a heritable morphological change. After 72 h culture in soft agar, colonies were found in B[a]P-treated HELFs. There were no colonies in DMSO control. It suggested that the B[a]P-treated cells were provided with some characteristics of transformed cellsin T-HELFs. Most of T-A-D1 cells and T-A-K4 cells were in G1 phase. It suggested that even without the stimulation of growth factor, T-HELFs could proliferate rapidly and the inhibition of cyclin D1 and CDK4 expressions blocked the cell cycle changes.In T-HELFs, the cyclin D1 protein level was significantly higher than that in HELFs (Fig. 4, lane 2). The expressions of E2F-1/4 and CDK4 did not change significantly, compared with HELFs. The expressions of cyclin D1 and CDK4 decreased significantly in T-A-D1 and T-A-K4 cells. Transfections of antisense cyclin D1 and antisense CDK4 increased the expression of E2F-4 (Fig. 4, lanes 3 and 4). These results showed that both cyclin D1 and CDK4 negatively regulated the expression of E2F-4 in T-HELFs.