Nicotine induces the fragile histidine triad methylation in human esophageal squamous epithelial cells

Authors


Abstract

The fragile histidine triad (FHIT) gene has been proposed to have an important role in very early carcinogenesis. Methylation of the FHIT gene is associated with transcriptional inactivation in esophageal squamous cell carcinoma, and FHIT inactivation has been linked to smoking-related carcinogenesis. In this study, we confirmed methylation of the FHIT gene in human esophageal squamous epithelial cells (HEECs) and examined whether nicotine induced alteration of FHIT. Methylation status in the promoter region of the FHIT gene and p16INK4A gene was determined by methylation-specific PCR in HEECs exposed to nicotine under various conditions. Methylation status of the FHIT gene was confirmed by DNA-sequencing analysis. Protein expression of Fhit and the DNA methyltransferases (DNMTs) DNMT1 and DNMT3a were assessed by immunoblot analysis. In the absence of nicotine, methylation of the FHIT gene and attenuation of Fhit protein were not detected in HEECs. Nicotine induced the methylation of FHIT gene and attenuated Fhit protein in association with increased expression of DNMT3a. Reexpression of Fhit protein in HEECs was found after cessation of moderate- to long-term exposure to nicotine. Our results show that nicotine induces methylation of the FHIT gene followed by loss of Fhit protein expression in HEECs. Continuous smoking may thus increase the risk of esophageal cancer. © 2006 Wiley-Liss, Inc.

The development of human cancer is generally considered a multistep process involving multiple genetic or epigenetic changes. Epigenetic changes such as aberrant DNA methylation are an alternative way to inactivate tumor suppressor genes (TSGs) and may have important roles in the development of esophageal cancer1, 2 DNA methylation patterns are established and maintained by 3 DNA methyltransferases (DNMTs): DNMT1, DNMT3a and DNMT3b.3, 4 DNMT1 has traditionally been regarded as a maintenance methyltransferase that specifically copies DNA methylation patterns after DNA replication.5 In contrast, DNMT3a and DNMT3b are implicated primarily in de novo methylation.6, 7

Recent studies have localized the fragile histidine triad (FHIT) gene at 3p14.2,8 considered a candidate TSG. FHIT is inactivated during the development of precancerous lesions of various types.9, 10 We have previously confirmed promoter methylation of the TSG p1611 and FHIT12 in esophageal squamous cell carcinoma (ESCC) cell lines established at our department.13 Pekarsky et al. suggested that FHIT inactivation occurs very early in the development of carcinogen-induced tumors. Carcinogens from cigarette smoke and other sources cause deletions in the FHIT locus, leading to the loss of Fhit protein expression. Then, cells lacking FHIT expression presumably acquire growth advantages, proliferate, alter p16, p53, or other genes, and form precancerous lesions.14 Therefore, inactivation of FHIT may be a downstream effect of defects in DNA repair machinery. Such inactivation may be a very early event in carcinogenesis, even preceding alteration of p16INK4A.14, 15 The etiology of ESCC is strongly linked to the inactivation of FHIT or p16, as well as to cigarette smoking.16, 17 Considerable information on molecular abnormalities potentially related to cigarette smoking is now available. For example, FHIT gene abnormalities have been associated with a history of smoking in patients with malignant tumors,18 and tobacco smoking has been linked to promoter methylation in p1610 or FHIT.14

Nicotine is the major addictive agent in tobacco,19 and addicted individuals continue to smoke and use tobacco products to maintain plasma nicotine levels. Although studied extensively, nicotine has not been proven to be carcinogenic. However, researchers have long suspected that nicotine might promote tumor growth.20 Zhang et al. showed by microarray analysis that nicotine alters gene expression.21 However, whether nicotine is endogenously converted to more carcinogenic counterparts through a yet unidentified metabolic pathway remains unknown. In addition, it is unclear whether and, if so, how nicotine metabolism contributes to carcinogenesis in smokers.

This study examined FHIT gene methylation in human esophageal squamous epithelial cells (HEECs). We carried out a series of experiments to determine whether nicotine can cause de novo methylation of the FHIT gene and to examine whether FHIT gene methylation occurs before p16 gene methylation.

Material and methods

Cell culture

We used HEEC lines previously established in our laboratory.22 The HEECs were cultured from normal esophageal epithelium, obtained from resected specimens of the esophagus of a nonsmoking patient with esophageal carcinoma. Written informed consent was obtained from the patient regarding the performance of surgery and the use of resected samples for research. (The approval numbers of Kyoto University Institutional Review Board were 232 and G48, respectively.) Tissue samples were confirmed to be free of macroscopic evidence of tumor tissue as well as histological evidence of metaplastic cells and cancer cells. Cells were grown in keratinocyte-SFM supplemented with bovine pituitary extract and epithelial growth factor (Gibco BRL, Life Technologies, Rockville, MD) in a 5% CO2/water-saturated incubator at 37°C. Under these conditions, cells that were successfully passaged more than 15 times without senescence were defined to be “established cell lines.” We also used an ESCC cell lines that we had previously established, KYSE-150.13 In our previous study,12 we extracted DNA from KYSE-150 to serve as a methylated form of the FHIT gene. Since our HEEC lines were not available at that time, we used DNA from normal esophageal tissue to serve as the unmethylated form. In the present study, however, we were able to use HEEC cell lines instead of normal esophageal tissues. We therefore used DNA from KYSE-150 as positive control for the methylated form of the FHIT gene and HEECs as positive control for the unmethylated form.

Nicotine treatment

Cells were seeded at 3 × 106 cells/10-cm culture dish. After cell attachment (approx. 2 days), 0, 10 or 100 μM of nicotine (Sigma, St. Louis, MO) was added to the medium. Subsequently, we changed the medium every 4th day. When the cells became about 80% confluent, they were harvested and seeded at 3 × 106 cells/dish on new 10-cm culture dishes. The remaining cells were collected as samples. We freshly prepared nicotine dilutions with the use of nicotine-free base each time. The 100 μM concentration of nicotine used in this study is within the usual nicotine concentration in the saliva of smokers who average 25 cigarettes/day.23

Methylation analysis

We collected sample cells from the dishes by using trypsin/EDTA. The cells were counted by the trypan blue dye-exclusion method. Next, DNA was prepared from the collected cells by using a DNA extraction kit (Wako Pure Chemical Industries, Osaka, Japan). The methylation status in the promoter region of the FHIT gene and p16INK4A gene was determined by methylation-specific PCR (MSP), as described previously.24, 25 Briefly, 2 μg of genomic DNA was modified with sodium bisulfite. PCR amplification of bisulfite-modified DNA was performed with the use of previously described primers.24, 25 Negative control samples without DNA were included for each set of PCR. PCR products were analyzed on 15/25% gradient polyacrylamide gels (Daiichi Pure Chemicals, Tokyo, Japan) with ethidium bromide and visualized under ultraviolet illumination. The PCR reactions for all samples showing methylation were repeated to confirm the results. Furthermore, to confirm the efficiency of the bisulfite modification and the specificity of MSP, we sequenced the PCR products as described previously.26 Briefly, both the unmethylated and methylated products were excised from the gel and amplified by a TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA). The products were then purified with a S.N.A.P. MiniPrep Kit (Invitrogen). Multiple clones from each PCR product were sequenced by means of automated DNA sequencers (Shimadzu-Biotech, Kyoto, Japan).

Protein extraction

Protein extraction was performed as described previously.27 Briefly, cell dishes were washed twice with cold phosphate-buffered saline and scraped into 400 μL of lysis buffer. The cell lysates were immediately stored on ice and then sonicated. The supernatants were collected by centrifugation. The protein concentration of the supernatant was quantified with the use of Protein Assay Reagent (PIERCE, Rockford, IL). The supernatant was treated with SDS-reducing sample buffer, boiled for 5 min, and subjected to SDS-PAGE.

Analysis of protein expression

Samples of lysate containing 30 μg of protein were electrophoresed on 4–25% gradient polyacrylamide gels (Daiichi Pure Chemicals, Tokyo, Japan) and were transferred to a Hybond-PVDF membrane (Millipore, Bedford, MA). The membrane was blocked for 1 hr at room temperature in a blocking buffer (5% fetal bovine serum). Primary and secondary antibodies were diluted in blocking buffer. Primary antibodies were incubated overnight at 4°C with a 1:1,000 dilution of the rabbit antibody against human Fhit (Zymed Laboratories, San Francisco, CA) and a 1:200 dilution of the rabbit antibody against human Dnmt 1 and 3a (Santa Cruz Biotechnology, Santa Cruz, CA). A secondary antibody solution (1:2,000 dilution) containing anti-rabbit immunoglobulin labeled with horseradish peroxidase (Zymed Laboratories, San Francisco, CA) was applied for 1 hr at room temperature. Membranes were treated with ECL detection reagents (Amersham Pharmacia, Piscataway, NJ), following the manufacturer's instructions. Then, the membrane was stripped under conditions recommended by the manufacturer, blocked and reprobed with β-actin (Sigma) to serve as control for uniformity of loading. Protein expression was quantified on a Macintosh computer using the public domain NIH Image program (developed at the US National Institutes of Health, available at http://rsb.info.nih.gov/nihimage/).

Results

Nicotine-induced methylation of the FHIT gene

First, we evaluated whether the FHIT gene of HEECs was methylated by continuous treatment with nicotine. Cells were divided into 3 groups and exposed to 10 μM of nicotine, 100 μM of nicotine, or no nicotine. The same conditions were maintained for 10 consecutive passages. We examined methylation of the FHIT gene by MSP. We also assessed protein expression of Fhit, DNMT1, and DNMT3a by immunoblot analysis. In the absence of nicotine, no methylation of the FHIT gene was detected, and expression of Fhit protein was strongly positive (Fig. 1a and 1b). No expression of DNMT3a protein was detected, whereas DNMT1 protein was constitutively expressed (Fig. 1b). In the presence of 10 μM of nicotine, no methylation of the FHIT gene was detected (Fig. 1a); however, in the presence of 100 μM of nicotine, methylation of the FHIT gene and consequent attenuation of Fhit protein expression were detected (Figs. 1a and 1b). DNMT1 protein expression was detected on the first passage after treatment with nicotine had begun, but was inhibited by continuous treatment with nicotine (100 μM) after the fifth passage (Fig. 1b). Exposure to nicotine (100 μM) caused stable DNMT3a protein expression (Fig. 1b). The unmethylated FHIT gene was observed in all samples throughout the experiment (Fig. 1a).

Figure 1.

Nicotine-induced methylation of FHIT gene of HEECs. DNA and protein from KYSE-150 and HEECs were extracted. Protein from KYSE-150 was used as positive control for the methylated form, and protein from HEECs was used as positive control for the unmethylated form (extreme right of 1a and extreme right of 1b). (a) Results for methylation of the FHIT gene of HEECs either exposed or not exposed to nicotine for 10 passages, as analyzed by methylation-specific PCR. The concentrations of nicotine used were 10 μM and 100 μM. 1a shows the results on the first, fifth and ninth passages. There was no appreciable methylation of the FHIT gene in HEECs without nicotine treatment (1a, left lane). Treatment with 10 μM nicotine also did not cause methylation of the FHIT gene (1a, middle lane). However, increasing the nicotine concentration to 100 μM resulted in methylation of the FHIT gene, and methylated status was maintained during continuous exposure to 100 μM nicotine (1a, right lane). M-FHIT, methylated FHIT; U-FHIT, unmethylated FHIT. (b) Effects of exposure to 100 μM nicotine for 10 passages on expression of Fhit, DNMT1, and DNMT3b protein were analyzed by immunoblot analysis. 1b shows the results of the first, fifth, and ninth passages. Fhit protein was well expressed without nicotine treatment, whereas continuous exposure to nicotine resulted in attenuation of Fhit protein expression. There was also constitutive expression of DNMT1 protein without nicotine treatment, which was inhibited by nicotine stimulation. There was no DNMT3a protein expression in the absence of nicotine treatment; stable DNMT3a protein expression was confirmed in the presence of nicotine.

Effects of short-term exposure to nicotine on methylation status

Next, we investigated when the gene was methylated by nicotine, and whether the methylated gene disappeared. We were able to grow HEECs for 8 days before they became confluent. The cells were then divided into 3 groups and were grown in the absence of nicotine, in the presence of 10 μM of nicotine and 100 μM of nicotine for the first 4 days followed by the absence of nicotine for the next 4 days. Similar to that indicated in Figure 1, there was no methylation of the FHIT gene in the absence of nicotine for 8 days (data not shown), whereas nicotine treatment for 8 days induced methylation of the FHIT gene in HEECs (data not shown). After the discontinuation of nicotine treatment, methylated FHIT was detected for 3 consecutive days (1* in lane 5 to 3* in lane 7, Fig. 2a), but disappeared on day 4 (4* in lane 8, Fig. 2a). Consistent with these results, Fhit protein expression was weak for 3 days (1* in lane 5 to 3* in lane 7, Fig. 2b), but strong on day 4 (4* in lane 8, Fig. 2b). DNMT3a protein was expressed during nicotine treatment (lanes 1–4, Fig. 2a), but its expression decreased after the cessation of nicotine treatment (lanes 5–8, Fig. 2a). In contrast, DNMT1 protein was still expressed 1 day after switching from a medium with nicotine (100 μM) when compared with that without nicotine (lanes 5, Fig. 2a), but its expression decreased on day 2 and eventually disappeared (lanes 6 to 8, Fig. 2a). To confirm whether methylation during very short exposure to nicotine was specific to the FHIT gene, we examined p16INK4A, a site frequently methylated in malignancies.25 There was no evidence of methylation of the p16INK4A gene in HEECs exposed to nicotine for 4 days (Fig. 2a). Hence, methylation of the FHIT gene and attenuation of Fhit protein expression were associated with short-term exposure to nicotine, but disappeared after the cessation of exposure.

Figure 2.

Effects of short-term exposure to nicotine on methylation of the FHIT gene and p16INK4a gene and Fhit protein expression. Figure shows methylation (a) and protein expression (b) in the presence of nicotine (100 μM) for 4 consecutive days, followed by the absence of nicotine for the next 4 days. We collected cell samples daily for 8 days. KYSE-150 served as positive control for the methylated form, and HEECs served as positive control for the unmethylated form (extreme right, 2a and extreme right, 2b, respectively). *, Days after changing the culture medium from the presence to the absence of nicotine (100 μM). (a) Methylation of both the FHIT gene and p16INK4a gene of HEECs was analyzed by methylation-specific PCR. M-FHIT, methylated FHIT; U-FHIT, unmethylated FHIT; M-p16, methylated p16; U-p16, unmethylated p16. After removal of nicotine (100 μM) from the medium, methylated status continued for 3 days (1* in lane 5, to 3* in lane 7), but not on day 4 (4* in lane 8). (b) Fhit, DNMT1 and DNMT3b protein expression of HEECs after treatment with nicotine (100 μM) for 4 days followed the absence of nicotine for next 4 days, as analyzed by immunoblot analysis. After removal of nicotine (100 μM) Fhit protein was not expressed for 3 days (1* in lane 5 to 3* in lane 7), but strong expression of Fhit protein was detected on day 4 (4* in lane 8).

Effects of moderate- to long-term exposure to nicotineon methylation status

Next, we evaluated the effects on gene methylation of moderate- to long-term exposure of HEECs to nicotine. We maintained HEECs for 8 passages (12 weeks). As described earlier, cells were divided into 3 groups and grown in media with nicotine (100 μM), without nicotine, or with nicotine (100 μM) for the first 4 passages and without nicotine for the next 4 passages. We confirmed no methylation of the FHIT gene during any of the 8 passages in the absence of nicotine and persistent methylation of the FHIT gene during all 8 passages in the continuous presence of nicotine (data no shown). After switching from a medium with nicotine (100 μM) to one without nicotine, methylation of the FHIT gene continued for 3 passages (lanes 5–7, Fig. 3a); demethylation of the FHIT gene apparently occurred on the fourth passage (lane 8, Fig. 3a). Consistent with these findings, Fhit protein expression was weak during the first 3 passages after switching to a nicotine-free medium (lanes 5–7, Fig. 3b), but was strong on the fourth passage (lane 8, Fig. 3b), seeming to follow demethylation of the FHIT gene. Methylation of the FHIT gene and absence of Fhit protein expression were caused by moderate- to long-term exposure to nicotine, but were reversed by the cessation of nicotine.

Figure 3.

Effects of moderate- to long-term exposure to nicotine on methylation of the FHIT gene and Fhit protein expression of HEECs. The figure shows methylation (a) and protein expression (b) patterns in the presence of nicotine (100 μM) for 4 passages, and then in the absence of nicotine for the next 4 passages. Protein from KYSE-150 was used as positive control for the methylated form, and protein from HEECs was used as positive control for the unmethylated form (extreme right, 3a and extreme right, 3b, respectively). (a) Effects of moderate- to long-term exposure to nicotine on FHIT of HEECs. Methylation of the FHIT gene of HEECs was analyzed by methylation-specific PCR. The nicotine concentration used was 100 μM. HEECs were cultured in the presence of nicotine (100 μM) for the first 4 passages; nicotine was then removed from the medium. After cessation of nicotine exposure, methylated status was maintained for 3 passages (1* in lane 5 to 3* in lane 7), but disappeared on the fourth passage (4* in lane 8). (b) Reexpression of Fhit protein of HEECs after cessation of moderate- to long-term exposure to nicotine. Fhit protein expression of HEECs was analyzed by immunoblot analysis. After removal of nicotine, lack of Fhit protein expression continued for 3 passages (1* in lane 5 to 3* in lane 7), but strong expression of Fhit protein was detected on the fourth passage (4* in lane 8). *, Passages after removal of nicotine (100 μM) from the medium.

Genomic sequencing of PCR products

We used sequencing to confirm the methylation status of methylated PCR products. Genomic sequencing of methylated samples after bisulfite treatment of DNA and MSP confirmed that cytosine residues were replaced by thymine residues, while 5′-methylcytosine residues were unaffected by the treatment. The frequency of methylation was consistently about 36% of the total cytosine residues at any given point.

Discussion

Promoter methylation may constitute the first hit in cancer, and methylation involving the second gene copy often constitutes the second hit, eliminating the remaining gene copy. Inactivation of FHIT occurs very early in the development of cancer. Biallelic inactivation of the FHIT gene can result from epigenetic inactivation and be reversed by exposure to 5-azacytidine.24 Although inactivation of other TSGs is probably required for carcinogenesis, inactivation of FHIT may be an important step in the development of many cancers.15, 28 This process might be accelerated by cigarette smoking, which increases DNMT expression,29 potentially promoting gene methylation.

Our results showed that methylation of FHIT occurred on exposure to nicotine, whereas demethylation occurred after cessation of nicotine. Continued treatment with nicotine maintained the methylation status of the FHIT gene. Expression of Fhit protein consequently decreased on exposure to nicotine. Demethylation of the FHIT gene after discontinuation of nicotine exposure led to the reexpression of Fhit protein. These findings suggested that the FHIT gene of esophageal mucosal cells may be directly or indirectly inactivated by exposure to nicotine. Moreover, we speculated that nicotine-induced methylation of the allele and then caused changes that inactivated other alleles, thus leading to gene inactivation. In contrast, the p16INK4A gene showed no methylation after short-term exposure to nicotine. It was unclear why FHIT was affected, while p16INK4A was not. We believe that both the FHIT and p16INK4A genes are affected (methylated) by nicotine, but methylation of the FHIT gene occurs before methylation of p16INK4A, perhaps because the former is more sensitive to nicotine treatment. Our results support this possibility. We also showed that a longer exposure time to nicotine was associated with delayed demethylation of the FHIT gene. We have previously reported that Fhit protein expression is associated with cancer progression, but may not be related to patients' outcomes or smoking histories.30 Available evidence suggests that FHIT gene modification by cigarette smoking might have a role in precancerous conditions early in the development of cancer, but may not have a clinically significant role in later stages, after the establishment of cancer.

Our results suggest that even concentrations of nicotine in the saliva of persons receiving nicotine replacement therapy, estimated to be below 100 μM,31 merit concern. Long-term nicotine replacement therapy to maintain cessation of smoking might carry an increased risk of cancer.32

Finally, our results provide evidence that nicotine activates expression of the de novo DNA methyltransferase DNMT3 to induce methylation of the FHIT gene. Discontinuation of nicotine exposure was associated with demethylation of the once methylated FHIT gene. Our findings also imply that continuous nicotine treatment may maintain the methylation status of the FHIT gene. At present, the mechanism involved remains unclear. Furthermore, we could not explain the mechanism underlying the 1-day time lag before the change in DNMT1 expression after cessation of exposure. These and other related topics should be addressed in future studies.

In conclusion, our results suggest that the cessation of smoking may reduce the risk of esophageal cancer. Among many factors potentially involved, continued expression of Fhit protein may have an important role in risk reduction. Earlier cessation of smoking is thus likely to be of greater benefit in terms of cancer prevention.

Acknowledgements

Supported in part by Grant-in-Aid 14370385 (to Y.S.) from the Japanese Ministry of Education, Culture, Sports, Science and Technology.

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