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Cell Biology International (2006) 30, 183–189 (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


Abstract

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.


Keywords: All-trans retinoic acid, Benzo(a)pyrene, Cyclin D1, CDK4, E2F, Cell cycle.

*Corresponding author. Tel./fax: +86 10 8313 2527.


1 Introduction

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 48–72h.

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 (G1) requires cyclin D-dependent kinase(CDK4 and CDK6) and cyclin E/CDK2 activity. The D-type cyclins (cyclins D1, D2, and D3) complex with CDK4 and CDK6 and thereby regulate transition from the G1 phase into the S phase by phosphorylation and inactivation of pRb and release of sequestered E2F transcription of genes encoding proteins that are required for S-phase DNA synthesis, promoting entry into S phase (Sherr and Roberts, 1995). Some investigators have shown that cyclin D1 is frequently overexpressed in a variety of other human carcinomas (Suzui et al., 2002), and RA reduced cyclin D1 and CDK expression in human hepatomas cells and human coronary smooth muscle cells (Wakino et al., 2001; Suzui et al., 2002).

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

2.1 Chemicals

B(a)P, DMSO, ATRA and l-glutamine were purchased from Sigma. Both ATRA and B(a)P were dissolved in DMSO to produce stock solution of 2mM and 5mM, respectively. The terminal concentration of working solution were ATRA 0.1, 1 and 2μM and B(a)P 2μM, which were stored at −20°C to avoid photoisomerization. The concentration of DMSO in cell culture medium never exceeded 0.1%. RPMI-1640 was purchased from GIBCO, and gentamycin sulfate was from Amresco. Antisense cyclin D1 plasmid and antisense CDK4 plasmid were provided by Dr. Yan (Yan et al., 2003).

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 2.0g NaHCO3 per liter, 10% fetal bovine serum (FBS), 2mM l-glutamine (Sigma), 50μg/ml gentamycin sulfate (Amresco), in an incubator with humidified atmosphere at 37°C with 5% CO2 in air. Cells were serum starved for 24h prior to the pretreatment with ATRA at a final concentration of 0.1, 1 and 2μM. Fresh culture medium was replenished every 24h avoiding ATRA own degradation, after ATRA treatment for 24h, B(a)P was supplied at final concentration of 2μM for 24h. All the treatments were maintained at 37°C with 5% CO2 in air. As an untreated solvent control, cells were treated with DMSO not exceeding 0.1%. All handling with ATRA and B(a)P was performed under subdued light, and flasks containing ATRA were covered with aluminum foil.

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 (10μl) and antisense cyclin D1 plasmid (2μg) or antisense CDK4 plasmid (2μg) to transfect antisense cyclin D1 and antisense CDK4 into HELF respectively in the absence of serum. After 2h, the medium was replaced with RPMI-1640 containing 10% FBS. Approximately 48h after the transfection, the cells were dissociated with 0.25% trypsin and cell suspensions were plated into 75-ml culture flasks and cultured for 14days with G418 selection (400μg/ml). The stable transfectants, HELF antisense cyclin D1 or HELF antisense CDK4, were established and cultured in G418-free RPMI-1640 for at least two passages before each experiment.

2.4 Western blot analysis

HELF cells were lysed with cell extraction buffer, which contained 50mM Tris–HCl (pH 6.8), 100mM DTT, 2% SDS, 0.1% bromophenol blue, and 10% glycerol. Total protein was separated on 5–10% SDS–polyacrylamide gels by electrophoresis and transferred to nitrocellulose membranes (Bio-Rad). After incubation at 4°C overnight with primary antibody including actin, cyclin D1, CDK4, E2F-1 or E2F-4, corresponding secondary antibodies were supplied for 1h at room temperature, respectively. Protein was visualized using ECL (enhanced chemiluminescence) detection system (Amersham). Each blot was quantitated by digital scanning.

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 24h prior to the treatment ATRA at a final concentration of 2μM; after 24h, B(a)P was supplied at a concentration of 2μM for another 24h. Cells were dissociated with trypsin and washed in cold phosphate-buffered saline, and fixed with 70% cold ethanol on ice for 30min. The suspensions were centrifuged at 1500rpm for 5min. The pellet was resuspended in a solution containing 50μg/ml propidium iodide, 1mg/ml sodium citrate, 0.3% Nonidet P40 and 5μg/ml RNase A, and stained on ice for at least 40min. Then the pellets were analyzed by a flow cytometer.

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<0.05 considered to be significant.

3 Results

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 2μM B(a)P could alter the expression of cyclin D1 and CDK4, the G1 cell cycle control proteins. We also examined the expression of E2F-1 and E2F-4, and found that after HELF were treated with 2μM B(a)P for 24h, there was a marked increase in the cyclin D1 and E2F-1 expression (Fig. 1A,B). There were almost no changes of CDK4 (Fig. 1A) and E2F-4 (Fig. 1A) expression.


Fig. 1

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 2μM) for 24h, B(a)P was supplied at final concentration of 2μM for 24h. The cells were lysed for Western blot analysis. (B) Quantification analysis of Western blots for cyclin D1 and E2F-1, as shown in (A). Results represent three independent experiments. Data were analyzed using one-way ANOVA. *P<0.05 compared with group 1.


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 2μM B(a)P, a dose–response experiment was done with ATRA at 0.1μM, 1μM and 2μM. After serum starvation for 24h, cells were treated with different concentrations of ATRA. One day later 2μM B(a)P was supplied for a further 24h.

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.


Fig. 2

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 (0.1μM) for 24h, B(a)P was supplied at final concentration of 2μM for 24h. The cells were lysed for Western blot analysis. (B) Increased percentage of cyclin D1 and E2F-1 expression in HELF and HELF transfected with antisense cyclin D1 and CDK4 after treatment with B(a)P (2μM). (C) Decreased percentage of cyclin D1 and E2F-1 expression in HELF and HELF transfected with antisense cyclin D1 and CDK4 after pretreatment with ATRA (0.1μM). Data were analyzed using one-way ANOVA. *P<0.05 compared with HELF.


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 G1 phase after serum starvation for 72h (Table 1). Quiescent HELF cells were induced to enter S phase by stimulation with B(a)P and the population of cells in G1 phase decreased substantially (Table 1). ATRA inhibited G1–S progression, as reflected by the higher percentage of G1 phase cells (Table 1). The B(a)P-induced cell cycle progression from G1 phase to S phase was slower in antisense cyclin D1 stable transfectants than in similarly treated HELF (Fig. 3A). The same trend was found in antisense CDK4 stable transfectants (Fig. 3A). The ATRA-induced increase of the population of cells in G1 phase was slower in antisense cyclin D1 and CDK4 stable transfectants than in similarly treated HELF (Fig. 3B). These parallel studies further suggested that ATRA inhibited B(a)P-induced cycle progression partly through the cyclin D1/E2F-1 pathway in HELF.


Table 1.

Effects of ATRA on B(a)P-induced cell cycle changes in HELF


Fig. 3

Effects of ATRA on B(a)P-induced changes of the percentage of HELF and HELF transfected with antisense cyclin D1 and CDK4 in G1 phase. (A) Decreased percentages of HELF and HELF transfected with antisense cyclin D1 and CDK4 in G1 phase after treatment with B(a)P (2μM). (B) Increased percentages of HELF and HELF transfected with antisense cyclin D1 and CDK4 in G1 phase after pretreatment with ATRA (0.1μM). Data were analyzed using one-way ANOVA. *P<0.05 compared with HELF.


GroupPercentage G1 phase cells (mean ± SE)
HELF + DMSO62.43 ± 4.91
HELF + B(a)P (2 μM)40.87 ± 3.87*
HELF + B(a)P (2 μM) + ATRA (0.1 μM)57.23 ± 1.25


4 Discussion

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 G1 to S phase. Inhibition of cyclin D1 could slow down the B(a)P-induced cell cycle progression from the G1 phase to S phase, as reflected by the higher percentage of G1 phase cells in HELF. Similarly, inhibition of CDK4 also inhibited the B(a)P-induced cell cycle progression by accumulation of cells in the G1 phase. Transition from G1 to S phase is mainly regulated by cyclin D1/CDK4 and cyclinE/CDK2 complexes; thus it is suggested that the overexpression of cyclin D1 and E2F1 might partly account for the B(a)P-induced cell cycle progression in HELF.

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 G1 phase. Both inhibition of cyclin D1 and inhibition of CDK4 can slow the B(a)P-induced cell cycle progression from the G1 phase to S phase as described above. On pretreatment with ATRA, the increased percentage of HELF transfected with antisense cyclin D1 and CDK4 in G1 phase was less than in similarly treated HELF. The changes of cell cycle progression were consistent with the changes of the cyclin D1 and E2F-1 expression by pretreatment with ATRA before stimulation with B(a)P, which further confirmed that ATRA blocked B(a)P-induced cell cycle progression partly through the cyclinD1/E2F-1 pathway.

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.

Acknowledgements

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 2005

doi:10.1016/j.cellbi.2005.08.014


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)