Expression of 15-lipoxygenase-1 is regulated by histone acetylation in human colorectal carcinoma

Hideki Kamitani1,2, Seijiro Taniura1, Hiroshi Ikawa1, Takashi Watanabe2, Uddhav P. Kelavkar3 and Thomas E. Eling1,4

1 Laboratory of Molecular Carcinogenesis, National Institutes of Environmental Health Sciences, Research Triangle Park, NC 27709, USA,
2 Division of Neurosurgery, Institute of Neurological Sciences, Tottori University School of Medicine, 36-1 Nishi-cho, Yonago 683-0805, Japan and
3 Renal Division and Glomerulonephritis Center, Emory University, Atlanta, GA 30322, USA


    Abstract
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 Abstract
 Introduction
 References
 
15-Lipoxygenase-1 (15-LO-1) is expressed at higher levels in human colorectal tumors compared with normal tissue. 15-LO-1 is expressed in cultured human colorectal cells, but only after treatment with sodium butyrate (NaBT), which also stimulates apoptosis and cell differentiation. We examined the regulation of 15-LO-1 in human tissue and the colorectal carcinoma cell lines Caco-2 and SW-480 by treatment with histone deacetylase (HDAC) inhibitors: NaBT, trichostatin A (TSA) and HC toxin. Northern and western analysis showed that expression of 15-LO-1 was up-regulated by these HDAC inhibitors. Furthermore, HDAC inhibitors stimulated promoter activity of the 15-LO-1 gene ~12-to 21-fold using the –331/–23 region of the 15-LO-1 promoter, as measured with a luciferase–15-LO-1 promoter–reporter system, suggesting that 15-LO-1 is regulated at the transcriptional level by HDAC inhibitors. Histone proteins in colorectal cells were acetylated after treatment with HDAC inhibitors. Histone acetylation was also measured in human colorectal tissue and a correlation was observed between increased histone acetylation and 15-LO-1 expression. Thus, regulation of 15-LO-1 expression in colorectal tissues appears to occur by a novel and new mechanism associated with histone acetylation. Moreover, these results suggest that 15-LO-1 is a marker that reflects histone acetylation in colorectal carcinoma.

Abbreviations: FBS, fetal bovine serum; H4, histone 4; HDAC, histone deacetylase; 15-LO-1, 15-lipoxygenase-1; NaBT, sodium butyrate; TSA, trichostatin A.


    Introduction
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 Abstract
 Introduction
 References
 
Reticulocyte-type 15-lipoxygenase (15-LO-1) oxidizes C18 fatty acids such as linoleic acid (1), lipoproteins (2) and also more complex substrates such as biomembranes (3). 15-LO-1 is expressed during distinct stages of reticulocyte development (4), in macrophages of atherosclerotic lesions (5) and in the eye lens during organelle degradation (6). These findings suggest that expression of 15-LO-1 is associated with maturation, senescence, membrane degradation and atherosclerosis. We recently demonstrated expression of 15-LO-1 in human colorectal carcinoma tissue (7) and in human colorectal carcinoma Caco-2 cells during sodium butyrate (NaBT)-induced apoptosis and cell differentiation (8). Understanding the regulatory mechanism for 15-LO-1 gene expression may provide clues to elucidate the biological role of 15-LO-1. The regulation of 15-LO-1 expression is poorly understood but it is known that IL-4 and IL-13 up-regulate 15-LO-1 in monocytes (9) and airway cells (10) through the STAT-6 pathway (11). However, the regulatory mechanism for induction of 15-LO-1 by NaBT in Caco-2 cells (8) may be different. Butyrate is a short-chain fatty acid, a product of fermentation of luminal carbohydrates and is found in millimolar concentrations in the lumen of the intestinal epithelium (12). NaBT induces hyperacetylation of histones by inhibiting histone deacetylation and is pharmacologically categorized as a histone deacetylase (HDAC) inhibitor. Recently there has been considerable interest in transcriptional regulation by histone acetylation. For example, HDAC inhibitors modulate the expression of several genes, such as {gamma}-globin (13), p21WAF1 (14), gelsolin (15) and myb (16) in cultured cells

To examine whether expression of 15-LO-1 is regulated by histone acetylation, expression of 15-LO-1 was measured by northern analysis after treatment of the colorectal carcinoma cell lines Caco-2 and SW-480 for 24 h with the HDAC inhibitors NaBT, trichostatin A (TSA) (17) and HC toxin (18). Cells grown under normal culture condition [fetal bovine serum (FBS)] did not express 15-LO-1, but NaBT, TSA and HC toxin induced 15-LO-1 expression in both cells (Figure 1AGo). The 2.7 kb band is reported to be the translated region and the 4.0 kb band is reported to contain the untranslated region (20). Additional bands observed between 2.7 and 4.0 kb in SW-480 cells are presumed to be the result of alternative splicing. Since hypermethylation in a promoter region of a gene can be related to HDAC activity (21), we measured expression of 15-LO-1 after treatment with the demethylating agent 5-azacytidine. However, no induction of 15-LO-1 was observed in either cell line treated with 500 nM 5-azacytidine (Figure 1AGo).



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Fig. 1. Induction of 15-LO-1 expression in Caco-2 and SW-480 cells by treatment with HDAC inhibitors. Caco-2 cells were grown in Eagle's minimal essential medium and SW-480 cells were grown in IMEM as described previously (8). (A) Northern blot analysis using a human 15-LO-1 specific probe of total cellular RNA (20 µg/sample). Lanes 1–5, Caco-2 cells treated for 24 h; lanes 6–10, SW-480 cells treated for 24 h. Lanes 1 and 6, FBS; lanes 2 and 7, 5 mM NaBT; lanes 3 and 8, 1 µg/ml TSA; lanes 4 and 9, 200 nM HC toxin; lanes 5 and 10, 500 nM 5-azacytidine (a demethylating agent). (B) Immunoblot analysis. Caco-2 and SW-480 cells were treated with HDAC inhibitors for 48 h and the proteins extracted. Expression of 15-LO-1 was estimated as described previously (8). Each lane contains 40 µg of total cell lysate and samples were separated on an 8% SDS–polyacrylamide gel. Lane 1, standard 15-LO-1 protein.

 
15-LO-1 protein expression was estimated after treatment with HDAC inhibitors by Western analysis using an antibody specific for 15-LO-1. The cells were cultured with or without HDAC inhibitors for 48 h, then harvested and lysates prepared. After treatment of Caco-2 and SW-480 cells with the HDAC inhibitors a 72 kDa immunoreactive band was detected in these cell lysates (Figure 1BGo).

Since 15-LO-1 expression was increased by HDAC inhibitors, we investigated whether HDAC inhibitors can stimulate activity of the 15-LO-1 gene promoter. Two different promoter–reporter constructs, –628/–23pGL2 and –331/–23pGL2, were used for this study (Figure 2AGo). We also used the pGL2-Basic reporter plasmid, which lacks the promoter region of 15-LO-1, as a negative control. The relative luciferase activity and fold activation by HDAC inhibitors were determined in Caco-2 and SW-480 cells incubated with each of the three HDAC inhibitors. Fold activation using the pGL2-basic reporter was <1.4 in Caco-2 cells (Figure 2BGo) and <1.6 in SW-480 cells (Figure 2CGo) after treatment with the inhibitors. On the other hand, 6.2-, 9.0- and 6.4-fold activation of the –628/–23pGL2 reporter was observed after incubation with NaBT, TSA and HC toxin, respectively, in Caco-2 cells, while 1.8-, 2.1- and 2.3-fold activation was detected after treatment of SW-480 cells with NaBT, TSA and HC toxin. Interestingly, stronger activation was detected by transfection of both cell lines with the –331/–23pGL2 reporter. Caco-2 cells showed 17-fold activation with NaBT, 21.3-fold with TSA and 19.5-fold with HC toxin, while SW-480 cells showed fold activations of 11.9 with NaBT, 19.9 with TSA and 19.1 with HC toxin. These observations indicate that the promoter region of 15-LO-1 is activated by the HDAC inhibitors in Caco-2 and SW-480 cells. In addition, either a distal region of the 5'-flanking region within 331 bp appears to be involved in transactivation of the 15-LO-1 gene by HDAC inhibitors or a repressor region is present upstream of 331 bp.



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Fig. 2. Stimulation of 15-LO-1 promoter activity in Caco-2 and SW-480 cells by HDAC inhibitors. (A) Human 15-LO-1 promoter–reporter constructs. Each fragment of the 5'-flanking region from –628 to –23 and from –331 to –23 was constructed in the pGL2-Basic luciferase reporter plasmid as –628/–23pGL2 and –331/–23pGL2, respectively (B and C). The constructed plasmids –628/–23pGL2, –331/–23pGL2 and pGL2-Basic in combination with pRL-null were transiently transfected into Caco-2 cells (B) and SW-480 cells (C) and luciferase activities were analyzed after 48 h treatment with FBS, 5 mM NaBT, 1 µg/ml TSA or 200 nM HC toxin. For transfection, cells were plated in 6-well plates at a density of 1x105 cells/well and incubated in FBS medium for 24 h. After washing the cells with phosphate-buffered saline, 1 mg luciferase reporter and 10 ng pRL-null internal control construct (Promega) were transfected using 5 ml of LipofectAMINE. The medium was replaced after 6 h with medium with and without the HDAC inhibitors and the cells incubated for 48 h. Luciferase activity is expressed as relative luciferase activity: firefly luciferase signal/ renilla luciferase signal. Fold activation by HDAC inhibitors is also calculated for each condition. Data shown are means ± SE (n = 3).

 
One of the prime pharmacological effects of HDAC inhibitors is an increase in acetylation of core histones (22). Moreover, accumulating evidence suggests that acetylation and deacetylation of histones play significant roles in the regulation of transcription in eukaryotic cells (23). To ascertain whether treatment with NaBT, TSA or HC toxin leads to acetylation of core histones in Caco-2 and SW-480 cells, acid-extractable proteins were analyzed (Figure 3Go). As reported previously (24), histone 4 (H4) exhibits the best resolution among histones. After FBS treatment H4 was in the unacetylated form in both Caco-2 and SW-480 cells and a single band was detected. Incubation with NaBT, TSA or HC toxin for 20 h led to a large increase in the tri- and tetra-acetylated forms of H4 and thus several additional bands were observed in the H4 region. This observation indicates that incubation with the HDAC inhibitors increased acetylation of histones in Caco-2 and SW-480 cells.



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Fig. 3. Histone acetylation in Caco-2 and SW-480 cells induced by HDAC inhibitors. The cell pellets were washed twice with 0.5 ml lysis buffer (10 mM Tris, pH 8.0, 13 mM EDTA) and resuspended in 0.4 N H2SO4. Cells were incubated on ice for 1 h, followed by centrifugation at 10 000 g for 5 min. Total histones were precipitated from the supernatant with 10x volumes of acetone at –20°C overnight. The precipitated histones were collected by centrifugation, dried and resuspended in distilled water. Histone acetylation was evaluated by fractioning the histones on acid/urea/polyacrylamide gels. The lower gel was 15% acrylamide, 2.5 M urea, 5% acetic acid with 0.5% TEMED and 0.15% ammonium persulfate. Gels were fixed and stained for 1 h in 0.25% Coomassie blue, 10% acetic acid, 40% methanol, then destained with repeated changes of acid/methanol. Cells were treated for 24 h with FBS, 5 mM NaBT, 1 µg/ml TSA or 200 nM HC toxin. The identities of the H4 bands were verified with standard H4 (lane 5). Acetylated H4 showed slower migration than unacetylated H4.

 
We next evaluated whether the histones are acetylated in colorectal tissues that express 15-LO-1. Expression of 15-LO-1 and acetylated H4 were measured by immunoblotting as shown in Figure 4Go. Seven surgical samples, four from adjacent normal tissues (lanes 1–4 in Figure 4Go) and three from colorectal carcinoma tissues (lanes 5–7 in Figure 4Go), from patients with colon carcinoma were examined. Total proteins and histone fractions isolated from FBS- and NaBT-treated Caco-2 cells served as negative and positive controls (lanes 8 and 9 in Figure 4Go). 15-LO-1 was expressed in the samples in lanes 1, 4–7 and 9, while acetylated H4 was detected in the samples in lanes 1, 5–7 and 9. Except for lane 4, which is adjacent normal tissue, 15-LO-1 expression and histone acetylation evaluated by detection of acetylated H4 appear to be linked. Moreover, the tissues with higher expression of 15-LO-1 also showed greatest expression of acetylated H4.



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Fig. 4. H4 acetylation and expression of 15-LO-1 in human colorectal carcinoma tissue. Surgically resected colorectal tumor samples and adjacent normal tissues were obtained from the University of North Carolina, Lineberger Comprehensive Cancer Center. Equal amounts (20 µg) of total protein isolated from samples were separated by 8% PAGE for analysis of 15-LO-1. Ten micrograms of histone from each sample were separated by 18% PAGE for analysis of acetylated H4. Anti-acetylated H4 antibody (Upstate Biotechnology) was used to detect the acetylated H4 antibody. Lane C, 15-LO-1 standard; lanes 1–4, normal tissues adjacent to the colorectal carcinoma; lanes 5–7, colorectal carcinoma tissues; lane 8, FBS-treated Caco-2 cells; lane 9, NaBT-treated Caco-2 cells.

 
15-LO-1 is highly regulated and its expression is restricted to specific mammalian cells and tissues. For example, expression of rabbit reticulocyte 15-LO is observed during erythroid cell differentiation (25) and is regulated by binding of hnRNP K and hnRNP E1 to the 3'-untranslated region of 15-LO mRNA. Other studies with normal undifferentiated human tracheal epithelial cells in culture showed that 15-LO-1 expression was only observed after differentiation to mucociliary epithelium (26). Likewise, the undifferentiated human colorectal carcinoma cells Caco-2 and SW-480 do not express 15-LO-1 unless the cells are stimulated to undergo apoptosis and cell differentiation, when 15-LO-1 expression is observed. Thus, expression is tightly regulated and appears to be associated with cell differentiation. Treatment of colorectal cells with HDAC inhibitors causes differentiation and apoptosis in Caco-2 (8) and SW-480 cells (data not shown) and increases expression of 15-lipoxygenase at the transcription level, suggesting a possible relationship between histone acetylation, regulation of 15-LO-1 expression and cell differentiation/apoptosis.

Histone acetylation is associated with transcriptional activity in eukaryotic cells (27). Recently the relationship between histone acetylation and transcription regulatory mechanisms has been clarified (23,24). Acetylation occurs at lysine residues on the N-terminal tails of histones, resulting in alterations in nucleosomal conformation, thus increasing the accessibility of transcription regulatory proteins to chromatin templates (28). It is unclear why a particular gene is transcriptionally regulated by histone acetylation. A specific transcription factor must be considered an important player in this regulation. The 5'-flanking region from –331 to –23 is important in activation of the 15-LO-1 gene by HDAC inhibitors in both Caco-2 and SW-480 cells (Figure 2Go). GATA-binding sites, a share-stress response element, (20) Sp1 and TATA sites are included in this region, but we could not identify the specific site within this region responsible for activation of 15-LO-1. There are a few reports about the involvement of a transcription factor in gene activation by HDAC inhibitors. The Sp1 site is responsible for activation of p21WAF/Cip1 in colon cancer cells after treatment with HDAC inhibitors (29) and in osteosarcoma cells (30). The distal CCAAT box and the 3'-flanking sequence (CCAATAGAC) are critical in induction of {gamma}-globin by HDAC inhibitors in human leukemia cells, as reported by McCaffrey et al. (13). However, none of the above reports showed a differential interaction between the critical promoter region and a particular transcription factor after treatment with HDAC inhibitors.

We have previously demonstrated an ~2- to 3-fold higher expression of 15-LO-1 in colorectal carcinomas as compared with adjacent normal tissues from cancer patients (7). The results from this study suggest a correlation between high expression of 15-LO-1 and expression of acetylated H4 (Figure 4Go), which was most apparent in carcinoma tissues. This finding may provide a clue as to why 15-LO-1 is highly expressed in human colorectal tissues, especially in the epithelial region. Short-chain fatty acids such as acetate, propionate, and butyrate are produced when dietary fiber is fermented by colonic bacteria (31). As a result, the colorectal epithelium is exposed to these short-chain fatty acids and 15-LO-1 expression occurs. However, why histones in carcinoma tissues are more highly acetylated than those in normal tissues is still unclear. Since 15-LO-1 expression correlates with histone acetylation, 15-LO-1 would be a suitable marker for colorectal carcinoma exposed to HDAC inhibitors such as butyrate. Because HDAC inhibitors stimulate cell cycle arrest (32) and apoptosis/cell differentiation (8), colorectal epithelial cells or tissues expressing 15-LO-1 could be a marker indicating cessation of proliferation in colorectal carcinoma. In contrast to our previous data (7), Shureiqi et al. (33) reported on increased expression of 15-LO-1 in normal colonic epithelium compared with paired colorectal carcinoma tissue by immunohistochemistry. The difference in 15-LO-1 expression between their samples and ours is not clear, but may depend on the extent of histone acetylation in each sample. Further investigation is required to clarify the importance of expression of 15-LO-1 in colorectal cancer. In conclusion, our data support the hypothesis that histone acetylation stimulated by HDAC inhibitors is strongly linked to expression of 15-LO-1 in colorectal tissues.


    Notes
 
4 To whom correspondence should be addressed Email: eling{at}niehs.nih.gov Back


    Acknowledgments
 
We thank Dr Benjamin Calvo (University of North Carolina, NC) for providing the human colorectal carcinoma samples. We also thank Mark Geller for his technical assistance and Drs Linda Hsi and Jennifer Nixon for their critical reading of the manuscript.


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Received August 8, 2000; revised October 17, 2000; accepted October 20, 2000.