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Cell Biology International (2006) 30, 183189 (Printed in Great Britain)
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
Xiaowei Jiaa, Bingci Liua*, Xianglin Shib, Ai Gaoa, Baorong Youa, Meng Yea, Fuhai Shena and Hongju Dua
aNational Institute of Occupation Health and Poison Control, Chinese Center for Disease Control and Prevention, 29 Nan Wei Road, Beijing 100050, China
bInstitute for Nutritional Sciences, SIBS, the Chinese Academy of Sciences, Shanghai 200031, China
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 G
Keywords: All-trans retinoic acid, Benzo(a)pyrene, Cyclin D1, CDK4, E2F, Cell cycle.
*Corresponding author. Tel./fax: +86 10 8313 2527.
The well-studied benzo(a)pyrene [B(a)P], a ubiquitous environmental pollutant of great concern, is a polycyclic aromatic hydrocarbon often present in food, such as charcoal-broiled food, cigarette smoke and petroleum byproducts, which can enter various regions of the respiratory tract appropriate for the size of the particles. Chronic, topical exposure to benzo(a)pyrene induces relatively high steady-state levels of DNA adducts in target tissues and alters kinetics of adduct loss (Talaska et al., 1996). Persons employed in aluminum reduction plants, coke ovens, and foundries may be exposed to B(a)P via inhalation. B(a)P is one of the most potent carcinogens to which humans are frequently exposed, and it initiates a range of toxic effects, including carcinogenesis, teratogenesis and impairment of the immune system (Ling et al., 2004; Fitzgerald et al., 2004; Sadikovic et al., 2004).
The vitamin A metabolite, all-trans retinoic acid (ATRA), and its other active derivatives are potent modulators of cell growth, differentiation, and apoptosis in a variety of cell types (Zheng et al., 1999; Wakino et al., 2001; Suzui et al., 2002; Crowe et al., 2003; Gumireddy et al., 2003; Nadauld et al., 2004). They can also be used as chemotherapeutic and chemopreventive agents in a variety of malignancies, such as leukemias, uterine leiomyoma, colon cancer, gastric cancer and breast cancer (Leder et al., 1990; Chen et al., 1991; Lallemand-Breitenbach et al., 1999; Gamage et al., 2000; Kelly et al., 2000; Kumar et al., 2001; Wu et al., 2002; Chun et al., 2003; Lango et al., 2003; Manor et al., 2003; Czeczuga-Semeniuk et al., 2004; Liu et al., 2004). Retinoic acid (RA) has also been suggested as efficacious in the treatment of lung cancer (Dahl et al., 2000; Chang et al., 2004). However, retinoid therapy is confounded by toxicity at pharmacological doses. In attempts to overcome these problems, numerous retinoids have been synthesized and studied over the past 20 years in search for compounds with higher efficacy and lower toxicity (Song et al., 2001; Andel and Rosier, 2004). ATRA regulated a variety of physiological processes and induced its own degradation within 48–72
At a molecular level, the actions of retinoids are primarily initiated by binding it to two families of nuclear receptor proteins: RARs and RXRs (each one including 3 different isotypes, α, β and γ), both members of the superfamily of ligand-dependent transcription factors, which are located in the promoter regions and interact with specific DNA response elements, called retinoic acid response elements (RARE) (Chu et al., 2003; James et al., 2003; Lanvers et al., 1998). RARs have transcriptional activation, functional domains for ATRA and DNA binding, and dimerization with other factors. The DNA binding domain contains two zinc finger motifs. RARs interact with cognate response elements in the promoters of many genes (Huang et al., 1997; Emionite et al., 2003).
Cyclins are key molecules in cell cycle control because of their specific and periodic expression during cell cycle progression. Different cyclin/CDK complexes are temporally activated at specific phases of the cell cycle. Progression through the first phase (G
These findings suggest that aberrant expression of cyclin D1 may play an important role in the development of B(a)P-induced cell cycle progression and RA may block B(a)P-induced cell cycle progression by cyclin D1 pathway. In this study using RNA transfection techniques, we investigated the pathway of inhibition of B(a)P-induced cell cycle progression by ATRA in HELF.
2 Materials and methods
B(a)P, DMSO, ATRA and
2.2 Cell lines and treatment with chemicals
HELF cell line was purchased from Peking Union Medical College. Cells were cultured in RPMI-1640 medium supplemented with 2.0
2.3 Generation of stable transfectants
HELF cells were cultured in 6-well plates. When they reached 85–90% confluence, each well was supplied with Transfetam Reagent (10
2.4 Western blot analysis
HELF cells were lysed with cell extraction buffer, which contained 50
2.5 Cell cycle analysis
Flow cytometry was performed to analyze cell cycle distribution. HELF were cultured in flasks with 10% FBS RPMI-1640. Cells were serum starved for 24
2.6 Statistical analysis
All data of Western blot and flow cytometry are shown as means with the standard error. Statistical analysis were performed using one-way ANOVA with the probability of P
3.1 B(a)P increased levels of cyclin D1 and E2F-1
First, Western blotting was carried out to determine whether treatment of HELF cells with 2
Effects of ATRA on B(a)P-induced changes of E2F-1/4, cyclin D1 and CDK4 expression in HELF. (A) After cells were treated with ATRA (0.1, 1 and 2
3.2 Pretreatment with ATRA blocked B(a)P-induced overexpression of cyclin D1 and E2F-1 in HELF
Since B(a)P can upregulate cyclin D1 and E2F-1 expression in HELF cells, we examined whether the pretreatment of HELF with ATRA was also through this pathway to block the B(a)P-induced changes. To find an effective concentration of ATRA to block the effect of 2
We next examined expression of cyclin D1, CDK4, E2F-1 and E2F-4 in HELF. The results showed that the overexpression of cyclin D1 and E2F-1 induced by B(a)P were both downregulated significantly by treatment with each concentration of ATRA in HELF (Fig. 1A,B). E2F-4 expression was not significantly changed (Fig. 1A). Although ATRA could inhibit B(a)P-induced cyclin D1 and E2F-1 overexpression, there was no significant effect on the protein expression observed by treatment of HELF with ATRA at these concentrations alone in HELF (Fig. 1A,B).
3.3 Treatment with antisense of cyclin D1 and antisense CDK4
To examine whether B(a)P upregulation of E2F-1 was in a cyclin D1-dependent manner in HELF and to determine whether altered cyclin D1 and CDK4 expression were sufficient to reproduce the effects of ATRA downregulation B(a)P-induced overexpression of E2F-1, we stably transfected antisense cyclin D1 and antisense CDK4 plasmids into HELF. The effects of antisense cyclin D1 and antisense CDK4 were assessed by examining the expression of cyclin D1 and CDK4, respectively. The results show that the expression of cyclin D1 and CDK4 were reduced by the expression of antisense cyclin D1 and antisense CDK4, respectively (Fig. 2A). Inhibition of cyclin D1 can significantly impair B(a)P-induced E2F-1 upregulation (Fig. 2A,B). As was shown with pretreatment with ATRA, cells expressing antisense cyclin D1 showed a lesser decrease of the B(a)P-induced overexpression of cyclin D1 and E2F-1 compared to similarly treated HELF (Fig. 2A,C). The E2F-4 expression was not changed significantly by the inhibition of cyclin D1 (Fig. 2A). Inhibition of CDK4 was able to impair the B(a)P-induced overexpression of E2F-1(Fig. 2A,B). The decrease of B(a)P-induced overexpression of E2F-1 was less in ATRA-pretreated antisense CDK4 stable transfectants than in similarly treated HELF (Fig. 2A,C). As a result of these studies, cyclin D1 might be an upstream signal that then regulates E2F-1 in HELF pretreated with ATRA before exposure to B(a)P. Inhibition of B(a)P-induced changes by ATRA may be partly through the cyclin D1/E2F-1 pathway.
Effects of ATRA on B(a)P-induced changes of E2F-1/4, cyclin D1 and CDK4 expression in HELF and HELF transfected with antisense cyclin D1 and CDK4. (A) After cells were treated with ATRA (0.1
3.4 Pretreatment with ATRA blocked B(a)P-induced cell cycle progression
Passage through the cell cycle is a highly regulated event, and a variety of safeguards have been incorporated into this process. These include regulation of the expression of cell cycle mediators (e.g. cyclins and Cdks), regulation of active kinase complexes (e.g. specific modulators of phosphorylation), and the expression of specific inhibitors of kinase complex activity. To determine whether there were alterations, we performed flow cytometry using propidium iodide (PI) staining. Confluent HELF accumulated in G
Effects of ATRA on B(a)P-induced changes of the percentage of HELF and HELF transfected with antisense cyclin D1 and CDK4 in G
B(a)P-induced cell cycle progression was inhibited by ATRA partly through the cyclin D1/E2F-1 pathway in HELF. The transcription factor E2F-1 is one target of cell cycle regulation. DNA binding and transcription mediated by E2F-1 is regulated by the retinoblastoma tumor suppressor protein (pRb), and the ability of pRb to bind E2F-1 is regulated by the CDK4/6-cyclin D activated kinase complex, which can hyperphosphorylate pRb and release it from E2F-1. These events are exquisitely synchronized within the cell cycle and are regulated by the availability of activated kinase complex.
There is increasing evidence that overexpression of cyclin D1 and E2F contribute to cell transformation and tumor development (Johnson et al., 1997; Zhang et al., 1997; Suzui et al., 2002). In previous studies, B(a)P was shown to modulate a wide variety of cellular processes (Talaska et al., 1996). At the molecular level, growth promotion mechanisms induced by B(a)P are not well known in various cells. We have now shown that B(a)P increased the expression of cyclin D1 and E2F1, the cell cycle regulator proteins. We sought to determine whether cyclin D1 is an upstream kinase involved in E2F-1 induction by B(a)P. We created suppression of cyclin D1 or CDK4 protein expression by using the antisense techniques (Sauter et al., 2000). Both inhibition of cyclin D1 and inhibition of CDK4 markedly impaired B(a)P-induced E2F-1 upregulation. The changes of E2F-1 expression were based on the changes of cyclin D1 and CDK4 in HELF treated by B(a)P. These studies suggest that cyclin D1 was an upstream kinase involved in E2F-1 induction by B(a)P in HELF. Our findings that the expression of cyclin D1 and E2F-1 were upregulated by B(a)P are similar to those in previous reports (Johnson et al., 1997; Zhang et al., 1997).
Our flow cytometry analysis showed that B(a)P could effectively promote HELF through cell cycle progression from the G
Numerous studies published recently have indicated that cyclin D1 and E2F were frequently deregulated by ATRA in various human cells (Lee et al., 1998; Sueoka et al., 1999; Suzui et al., 2002). In our study, pretreatment of HELF with ATRA could detectably downregulate the B(a)P-induced high levels of cyclin D1 and E2F-1, which are required for growth. To determine whether ATRA downregulated B(a)P-induced overexpression of E2F-1 in a cyclin D1-dependent manner in HELF, we used the antisense techniques. The decrease of B(a)P-induced overexpression of E2F-1 was less in ATRA-pretreated antisense cyclin D1 stable transfectants than in similarly treated HELF. In like manner, the B(a)P-induced overexpression of E2F-1 decreased less in ATRA-pretreated antisense CDK4 stable transfectants than in similarly treated HELF. It was suggested that ATRA downregulated B(a)P-induced E2F-1 overexpression in a cyclin D1-dependent manner in HELF. Our findings that ATRA can markedly decrease B(a)P-induced high levels of cyclin D1 and E2F-1 in HELF are similar to the data from some workers (Lee et al., 1998; Sueoka et al., 1999; Suzui et al., 2002), but different from others (Kosaka et al., 2001; Wakino et al., 2001; Wu et al., 2001) on the effects of ATRA on other cell types that are growth inhibited by ATRA. Similar to our findings in HELF, treatment with ATRA reduced the level of cyclin D1 in human bronchial epithelial cells and non-small cell lung cancer cells (Sueoka et al., 1999). However, contradictory results were reported by Kosaka et al. (2001), who found that the inhibition of vascular smooth muscle cell proliferation by treated with ATRA was correlated with decreased kinase activity of cyclin D3 and E, but not cyclin D1 kinase. Taken together, these results indicated that the target molecules for ATRA may vary among cell species.
In subsequent studies, the results of flow cytometry showed that pretreatment with ATRA before stimulation with B(a)P induced cell cycle inhibition, which was associated with arrest of the cells in G
Compared with E2F-1, E2F-4 was expressed without significant changes by pretreatment with ATRA before stimulation with B(a)P in HELF. Although we did not find marked changes of CDK4 expression by pretreatment with ATRA before stimulation with B(a)P in HELF in Western blot analysis, inhibition of CDK4 expression by antisense CDK4 can decrease B(a)P-induced overexpression of E2F-1. The reason for this may be due to the fact that treatment with B(a)P or pretreatment with ATRA before exposure to B(a)P could change CDK4 activity independently of protein expression level in HELF. It was reported that although the protein expression of CDK4 was not changed, the activity of CDK4 was reduced by ATRA in other cell systems (Wu et al., 2001). These hypotheses are worthy of our attention and we will study them in the future.
This work was supported by grants from the National Natural Science Foundation of China (30028019, 30371206), 973 National Key Basic Research and Development Program (2002 CB 512905), and Foundation of Institute for Nutritional Sciences (INS), USA.
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Received 14 June 2005/2 July 2005; accepted 10 August 2005doi:10.1016/j.cellbi.2005.08.014