Skip Navigation


Carcinogenesis Advance Access originally published online on January 12, 2006
Carcinogenesis 2006 27(5):1099-1104; doi:10.1093/carcin/bgi344
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
27/5/1099    most recent
bgi344v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (7)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Mulero-Navarro, S.
Right arrow Articles by Fernandez-Salguero, P.M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mulero-Navarro, S.
Right arrow Articles by Fernandez-Salguero, P.M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The dioxin receptor is silenced by promoter hypermethylation in human acute lymphoblastic leukemia through inhibition of Sp1 binding

S. Mulero-Navarro {dagger}, J.M. Carvajal-Gonzalez {dagger}, M. Herranz 1, E. Ballestar 1, M.F. Fraga 1, S. Ropero 1, M. Esteller 1 and P.M. Fernandez-Salguero *

Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Extremadura, Avenida de Elvas s/n, 06071-Badajoz, Spain and1 Cancer Epigenetics Laboratory, Molecular Pathology Programme, Spanish National Cancer Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain

* To whom correspondence should be addressed. Tel: +34 924 289422; Fax: +34 924 289419; Email: pmfersal{at}unex.es


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The transcription factor aryl hydrocarbon receptor (AhR) has relevant functions in cell proliferation. Interestingly, the AhR can either promote or inhibit proliferation depending on the cell phenotype. Although recent data reveal potential pathways for AhR signaling in cell proliferation, the mechanisms that regulate its activity in tumor cells remain unknown. Here, we have analyzed promoter hypermethylation as a potential mechanism controlling AhR expression in human tumor cells. AhR promoter CpG methylation was sporadic in a panel of 19 tumor cell lines except for the chronic myeloid leukemia (CML) K562 and the acute lymphoblastic leukemia (ALL) REH. When compared with normal lymphocytes, REH had very low constitutive AhR expression that could be attributed to promoter hypermethylation since treatment with the DNA demethylating agent 5-aza-2'-deoxycitidine (AZA) significantly increased AhR mRNA and protein. These results in leukemia-derived cell lines were further confirmed in primary ALL, where 33% of the patients (7/21) had AhR promoter hypermethylation. Chromatin immunoprecipitation (ChIP) showed that methylation impaired binding of the transcription factor Sp1 to the AhR promoter, thus providing a mechanism for AhR downregulation in REH cells. Therefore, promoter hypermethylation represents a novel epigenetic mechanism downregulating AhR activity in hematological malignancies such as ALL.

Abbreviations: AhR, aryl hydrocarbon (dioxin) receptor; ALL, acute lymphoblastic leukemia; AZA, 5-aza-2'-deoxicitidine; ChIP, chromatin immunoprecipitation; CML, chronic myeloid leukemia; FBS, fetal bovine serum; MSP, methylation-specific PCR; PCR, polymerase chain reaction


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Traditionally related to xenobiotic-induced toxicity and carcinogenesis (1), the aryl hydrocarbon (dioxin) receptor (AhR) is gaining relevance as regulator of endogenous cell functions. These include maintenance of liver homeostasis and vasculogenesis (2–4) and immune system maturation (5). Although the AhR contributes to cell cycle control through interaction with retinoblastoma (pRb) protein (6), its role on cell proliferation is controversial because it can either inhibit or promote cell cycle largely depending on the cell phenotype. For instance, whereas AhR activation induces growth arrest in Jurkat T cells (7), in T-cell leukemia (ATL) AhR overexpression is associated with leukemogenesis (8). In other cellular contexts, whereas ligand-dependent AhR activation results in growth inhibition of pancreatic (9) and prostate (10) cancer cells, downregulation of the unliganded AhR by small interfering RNA produces both an increase in proliferation of HepG2 liver and a decrease in growth of MCF-7 breast tumor cells (11). Nevertheless, the mechanisms that alter/modify the expression levels of the AhR gene in cancer cells are unknown. A potential mechanism could be provided by promoter hypermethylation.

Cytosine hypermethylation at CpG dinucleotides in the promoter of tumor suppressor genes represents a major mechanism for gene inactivation in cancer. Methylation at the 5' position of cytosine has been reported to alter or interfere with the correct binding of transcription factors to target sequences overlapping CpG dinucleotides (12–14), although it also has a positive effect in recruiting methyl-CpG binding activities that associate with histone deacetylases and other chromatin-modifying elements that lead to a transcriptionally silenced state (15).

Many genes become hypermethylated at their CpG islands-containing promoters and subsequently inactivated in human tumors of different ethiology (15,16). The pattern of aberrant hypermethylation is specific of the tumor type as it has been demonstrated for both cancer cell lines (17) and primary human tumors (18,19), suggesting that the specific loss of function derived from methylation contributes to malignant progression (20).

As mentioned above, a rather unknown aspect of AhR biology is how its expression is regulated in transformed cells. Here, we have analyzed promoter hypermethylation as a putative mechanism controlling AhR expression in human tumor cells. The results obtained indicate that AhR expression could be silenced by promoter hypermethylation in acute lymphoblastic leukemia (ALL) and points the AhR as a cell-specific inhibitor of cell proliferation and as a novel molecular marker of this disease. Further, these data support hypermethylation as a regulatory mechanism controlling the binding activity of transcription factors to DNA.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Human tumor cell lines and primary human tumors
The 19 human cancer cell lines analyzed were obtained from The American Type Culture Collection and the German Collection of Microorganisms and Cell Cultures. These included tumors of the breast (MCF-7, MDA-MB-453), colon (SW-480, HCT-116), lung (H-522), melanoma (SK-MEL-18), osteosarcoma (Saos-2, MG-63), rhabdomyosarcoma (A673), testis carcinoma (NTERA-2), choriocarcinoma (JEG-3), neuroblastoma (SK-N-SH), embryonic kidney (293-T), chronic myelogenous leukemia (SD-1), adult erythroleukemia (HEL-R), T-cell lymphoma (HUT-78), histiocytic lymphoma (U-937), acute lymphoblastic leukemia (REH) and chronic myeloid leukemia (CML) (K562). Cells were grown and maintained in the appropriate culture media supplemented with 10% FBS. Genomic DNA was demethylated by 72 h treatment with 1 or 2 µM AZA. AhR promoter methylation was also analyzed in 21 human primary ALL.

Analysis for a CpG island in the human AhR promoter
A 1550 nt fragment of the human AhR promoter, containing the transcription start site, was selected from the National Center for Biotechnology Information (NCBI) database using the accession number D31708 [GenBank] . This sequence was analyzed with the CpGPLOT software program (http://bioinfo.hku.hk/cgi-bin/) using the following settings: observed/expected CpG ratio of 0.6, minimum length island of 200 nt and minimum G+C content of 50%. A 702 nt fragment was identified that contained CpG islands and the transcription start site. Within this fragment, a 327 nt region was selected for further analysis.

DNA methylation analysis of the AhR
We determined AhR promoter methylation by polymerase chain reaction (PCR) analysis of sodium bisulfite-treated genomic DNA, which includes chemical conversion of unmethylated, but not methylated, cytosine to uracil, using two different procedures. First, methylation was analyzed by bisulfite genomic sequencing of the 327 nt CpG island of the human AhR promoter. Both strands of the template were sequenced using the following primers (with respect to the transcription start site): sense (–133/–114) 5'-ATGAGGGTGGGGTTTTTAC-3' and antisense (+194/+170) 5'-AAACTTCCTAAATCCAAAATACTTC-3'. Second, methylation-specific PCR (MSP) was performed using primers specific for the methylated or unmethylated (modified) DNA. The primers were (with respect to the transcription start site): unmethylated sense (+35/+54) 5'-GGTTGGGGAGTTTTGTTGAT-3' and unmethylated antisense (+173/+152) 5'-CTTCCCACCTACAAAACTCAAAC-3'; methylated sense (+35/+54) 5'-GGTTGGGGAGTTTCGTCGAC-3' and methylated antisense (+169/+152) 5'-CCGCCTACGAAACTCGAA-3'. A DNA sample from normal lymphocytes (NL) was used as negative control for the methylated allele. PCR products were applied onto non-denaturing 3% polyacrylamide gels that were stained with ethidium bromide and visualized under UV light.

RT–PCR
Total cellular RNA was isolated using TRIZOL reagent (Life Technologies: Carlsbad, CA, USA). Two microgram were reverse transcribed at 42°C for 60 min using oligo(dT) priming and SuperScript II reverse transcriptase (Gibco BRL: Carlsbad, CA, USA). The AhR was amplified using the following primers: forward, 5'-GGATTCTATGCCTTATACACA-3' and reverse, 5'-GCAGAGGTTAACATGATAGG-3'. To check for RNA integrity, GAPDH cDNA was amplified using the following primers: forward, 5'-TCTTCTTTTGCGTCGCCAG-3' and reverse, 5'-AGCCCCAGCCTTCTCCA-3'. Amplification was carried out for 35 cycles in 50 µl reaction mixture containing 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each dNTP, 0.5 µM each primer, 2.5 U Taq polymerase and an aliquot of each reverse transcription reaction. Cycling conditions were as follows: denaturation at 94°C for 1 min, annealing at 60°C for 1 min and extension at 72°C for 1 min. PCR products were resolved in 2% agarose gels and visualized by ethidium bromide staining.

Immunoblotting
Cells were collected by centrifugation and the pellet resuspended at 4°C in lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) containing protease inhibitors phenylmethylsulfonyl fluoride, leupeptin, aprotinin and pepstatin. After incubation on ice, lysates were clarified by centrifugation. Aliquots of 20 µg protein were mixed with SDS-sample buffer, denatured and electrophoresed on SDS–PAGE gels. Gels were transferred onto polyvinylidene difluoride membranes (Immobilon-P; Millipore: Billerica, MA, USA) that were blocked in Tris-buffered saline (50 mM Tris–HCl, pH 7.5, 150 mM NaCl and 0.1% Tween-20) containing 10% non-fat milk. Blots were incubated with anti-AhR antibody (Biomol: Playmouth Meeting, PA, USA) and washed in Tris-buffered saline. HRP-conjugated anti-rabbit IgG (Amersham: Uppasala, Sweden) was added to the membranes for 1 h at room temperature. After additional washing in Tris-buffered saline, Luminol reagent detection kit (Santa Cruz Biotechnology, CA, USA) was added and membranes were exposed to X-ray film.

Chromatin immunoprecipitation
To investigate in vivo interactions of Sp1 with the human AhR promoter, chromatin immunoprecipitation (ChIP) assays were performed as described previously (21). In brief, chromatin was fixed with 1% formaldehyde followed by lysis and shearing to an average length of 0.4–0.8 kb. Protein–DNA complexes were immunoprecipitated overnight at 4°C using 4 µg of Sp1-specific antibody (Santa Cruz Biotechnology). After eluting the DNA from the immunoprecipitates, PCR amplification was performed in 25 µl reaction mixture using specific primers for the AhR promoter. The sensitivity of the PCR amplification was evaluated on serial dilutions of total DNA collected after sonication (input fraction). PCR amplifications were carried out with 34 cycles at 94°C for 30 s, 60°C for 30 s and 72°C for 30 s. The amplified DNA was separated on 2% agarose gels and visualized with ethidium bromide. The AhR promoter was amplified with the following sets of primers: forward, 5'-CTCAAGGAAGACGGAATGGA-3' and reverse, 5'-CACGCTCTCGGAACAGA-3'.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Analysis for a CpG island in the human AhR promoter
By running a NCBI search, an entry for the human AhR gene was found (D31708 [GenBank] ) that contained upstream and downstream sequences relative to the transcription start site. The region of this sequence encompassing nucleotides –1017 to +533 was analyzed for CpG dinucleotides using the program CpGPLOT. After setting the confidence interval for CpG location (observed/expected of 0.6) and filtering the results by C+G content (60% minimum), a 702 nt CpG-rich island was found (–217 to +485, vertical lines) that contained the transcription start site (Figure 1A, +1). Within this CpG island, we selected a 327 nt sequence (–133 to +194) that contained 34 CpG dinucleotides susceptible to be methylated (Figure 1B, black boxes and Figure 1C, circles). Methylation of these cytosines was used to estimate the level of AhR promoter methylation in human tumor cell lines and primary tumors.


Figure 1
View larger version (36K):
[in this window]
[in a new window]
  		Fig. 1. Selection of a CpG island in the human AhR promoter and analysis of CpG methylation in human tumor cell lines. A 1550 nt fragment of the human AhR promoter was selected from the NCBI database and analyzed with the CpGPLOT software using the program settings indicated in Materials and methods. (A) A 702 nt CpG island (–217 to +485) was identified by the CpGPLOT software in the human AhR promoter that contained the transcription start site (+1). (B) A 327 nt fragment (–133 to +194) within this CpG island, containing 34 CpG dinucleotides (black boxes), was selected for methylation analysis. (C) The 327 nt region was amplified by PCR using specific oligonucleotide primers (underlined in B) and sodium bisulfite-treated genomic DNA from the cell lines indicated. PCR products were analyzed by automated fluorescence sequencing. Methylated cytosines in CpG dinucleotides (CpG) are indicated as close circles, whereas unmethylated cytosines (TpG) are indicated as open circles. (D) Representative sequences for the 327 nt region of the AhR promoter amplified by PCR using the primers indicated. Sequence from H-522 cells revealed sodium bisulfite-dependent C to T conversion at CpG dinucleotides (arrows). In REH cells, on the contrary, essentially all cytosines in CpG dinucleotides remained unmodified by sodium bisulfite treatment (arrows).

 
AhR promoter methylation in human tumor cells
Methylation status of each of the 34 CpG dinucleotides located within the –133/+194 island is shown in Figure 1C. Sodium bisulfite-treated DNA was purified from the indicated cell lines and the CpG island was amplified by PCR using the specific primers shown (Figure 1A, arrows). PCR products were sequenced in both strands and the consensus sequence for each CpG dinucleotide was plotted as unmethylated (open circles, TpG in either strand) or methylated (closed circles, CpG in both strands) cytosines (Figure 1C). AhR promoter methylation was minimal in non-lymphoid human tumor cells from the breast (MCF-7, MDA-MB-453), colon (SW-480, HCT-116), lung (H-522), melanoma (SK-MEL-18), osteosarcoma (Saos-2, MG-63), rhabdomyosarcoma (A673), testis carcinoma (NTERA-2), choriocarcinoma (JEG-3), neuroblastoma (SK-N-SH) and embryonic kidney (293-T). A panel of six lymphoid tumor cell lines was also analyzed and it was found that chronic myelogenous leukemia SD-1, adult erythroleukemia HEL-R, T-cell lymphoma HUT-78 and histiocytic lymphoma U-937 cells had an unmethylated AhR promoter. However, ALL REH and CML K562 had a large degree of methylation in the AhR promoter. For comparison purposes, DNA sequencing of a cell line having unmethylated AhR promoter (H-522) is shown together with the highly methylated REH in Figure 1D. Note the bisulfite-induced conversion of cytosine to thymine in CpG dinucleotides in H-522 but not in REH (vertical arrows). Thus, whereas the AhR promoter remained unmethylated in a variety of human tumor cell lines, it was heavily methylated in certain ALL and CML lymphoid cell lines.

Demethylation with AZA restored AhR mRNA and protein levels in highly methylated REH tumor cells
Among cytidine analogues, 5-aza-2'-deoxycitidine (decitabine, AZA) is the most commonly used demethylating agent in cultured cells. AhR mRNA level was markedly low in REH cells as compared with normal human lymphocytes (NL) (Figure 2A). To address whether low AhR expression in REH was due to promoter hypermethylation, treatments with AZA were performed. Indeed, AZA significantly restored AhR mRNA expression in REH cells in a concentration-dependent manner; for instance, at 1 or 2 µM, AhR mRNA increased to levels similar to those found in NL. Furthermore, AhR protein levels, which were also very low in REH cells, significantly increased after treatment with 2 µM AZA (Figure 2B).


Figure 2
View larger version (20K):
[in this window]
[in a new window]
  		Fig. 2. Treatment with AZA restored AhR mRNA and protein levels in REH cells. REH cells were grown and left untreated (0) or treated with 1 or 2 µM AZA. (A) Total RNA was isolated and analyzed for AhR expression by RT–PCR as indicated in Materials and methods. Normal human lymphocytes (NL) were used as a positive control to determine the physiological levels of the receptor. GAPDH mRNA expression was also analyzed to confirm RNA integrity and quantification. (B) REH cells were left untreated (0) or treated with 2 µM AZA, and total protein was extracted and AhR levels were analyzed by western immunoblotting using 15 µg protein and an AhR-specific antibody.

 
AhR promoter methylation in human primary ALL
On the basis of the hypermethylation status of the AhR promoter in the ALL cell line REH, a panel of 21 human primary ALL tumors were analyzed. MSP for the AhR revealed that 7 out of the 21 patients had promoter methylation, which indicates that this gene could be downregulated in around 33% of the human ALL. A representative set of 15 primary ALL is shown in Figure 3.


Figure 3
View larger version (12K):
[in this window]
[in a new window]
  		Fig. 3. The AhR was hypermethylated in human primary ALL. Genomic DNA was isolated from a panel of 21 human primary ALL and left untreated or treated with sodium bisulfite. MSP was performed using PCR primers specific for the unmethylated (U) or methylated (M) AhR allele. A negative control for the methylated allele was done using DNA from normal human lymphocytes (NL). A negative control for the PCR reaction was performed in the absence of DNA (H2O). The nomenclature for each primary ALL is indicated at the top of the figure. A representative polyacrylamide gel for 15 ALL patients is shown.

 
Hypermethylation blocked Sp1 binding to the human AhR promoter
An early characterization of the human AhR promoter revealed the absence of a canonical TATA-box and the presence of 6 GC-boxes potentially involved in Sp1 binding (22). Since these Sp1 binding sites (GGGCGG, located at –91/–86, –71/–66 and –50/–28 in /Figure 1B) overlapped methylated CpG dinucleotides in REH cells, and because methylation of Sp1 binding sites has been shown to regulate NF-1 expression, we have determined whether or not Sp1 binding to the AhR promoter was impaired in REH cells (Figure 4). ChIP assays demonstrated that a specific anti-Sp1 antibody immunoprecipitated a region of the human AhR promoter containing the 6 GC-boxes in H-522 cells, for which the AhR promoter is unmethylated, but not in REH cells, for which this promoter is methylated. Importantly, treatment with the demethylating AZA was able to restore AhR promoter immunoprecipitability by the anti-Sp1 antibody (REH AZA, Figure 4). These data strongly suggest that impairment of Sp1 binding to methylated GC-boxes could be responsible for AhR downregulation in REH cells.


Figure 4
View larger version (26K):
[in this window]
[in a new window]
  		Fig. 4. ChIP reveals that hypermethylation blocks Sp1 binding to the AhR promoter. Unmethylated H-522 or methylated REH leukemia cells were grown and left untreated or treated with 2 µM AZA. After fixation, cultures were processed for ChIP assays as described in Materials and methods. PCR was performed using AhR-specific oligonucleotides covering the promoter region that contains putative Sp1 binding sites (GGGCGG). Templates for PCR corresponded to the input used in the ChIP assay (input) and DNA obtained from immunoprecipitations performed in the absence (–Ab) or presence of anti Sp1-specific antibody (Sp1).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Much interest exists in understanding the role of the AhR in tumor development in the absence of xenobiotics, and the influence of cell phenotype on AhR-dependent tumor promotion is considered relevant and is supported by experimental evidence. Constitutive activation of AhR induced gastric tumorigenesis in the absence of xenobiotics (23) and it was associated with adult T-cell human leukemia (ATL) (8). In addition, AhR activation by exogenous ligands inhibited growth of human pancreatic cancer cells expressing high levels of receptor (9). Interestingly, however, constitutive AhR activation was also responsible for growth inhibition of Jurkat T cells (7) and thymic involution in transgenic mice (24). Except for the adverse association between the Arg554Lys polymorphism and soft tissue sarcoma (25), the mechanisms controlling AhR expression in human cancer are essentially unknown.

We have analyzed promoter hypermethylation as an epigenetic mechanism potentially regulating AhR expression in human tumors. Surprisingly, AhR promoter methylation was highly cell-specific, and out of 19 human tumor cell lines analyzed, representing 16 tumor types, only those from ALL (REH) and CML (K562) had significant AhR promoter hypermethylation. This result is in agreement with previous data showing that promoter hypermethylation is a cell-specific event, exhibiting wide variations between lymphoid and non-lymphoid tumor cells and also within similar lymphoid cell lines (18,26).

Promoter methylation accounted for lower endogenous AhR levels in REH cells since the demethylating agent AZA restored AhR mRNA and protein to levels similar to those present in normal human lymphocytes. Interestingly, a previous study has shown that the CML K562 cell line, found here to be hypermethylated, lacked AhR expression, whereas the U937, reported here to be unmethylated, had normal AhR levels (27). Considering the cell-specific promoter methylation pattern of AhR in ALL and CML, we have also analyzed a panel of 21 human primary ALLs. We have observed by MSP that 33% of the patients had AhR promoter hypermethylation. This frequency of hypermethylation is similar to those reported for other genes whose inactivation is considered relevant in ALL, such as p73 (30%) (28), p53 (32%) (29), p15 (40%) and p16 (8%) (30). Because these studies indicated that downregulation of tumor suppressor genes should be considered a relevant parameter in maintaining tumor cell proliferation and the pathogenesis of ALL, our results suggest that a decreased endogenous AhR level in ALL cells could be reflecting its inhibitory role in the control of cell proliferation.

The cloning and analysis of the human AhR gene promoter revealed the absence of a consensus TATA-box and the presence of binding sites for transcription factors such as the constitutively expressed Sp1 (22). However, the mechanisms regulating human AhR levels in normal or tumor cells remain mostly unknown. We report here that Sp1 could play a major role in maintaining constitutive AhR expression in human tumor cells. Furthermore, a plausible mechanism that could regulate endogenous AhR levels, at least in cancer cells, appears to involve differences in the methylation status of consensus sequences that favors or impairs transcription factors binding (e.g. Sp1) in a cell-specific manner. This hypothesis is supported by previous studies showing that NF-1 promoter hypermethylation decreased its levels by altering CREB and Sp1 binding (12).

In summary, our data suggest that promoter hypermethylation could represent a mechanism regulating AhR expression in human tumor cells in a cell-specific manner and reveals a novel example of how hypermethylation at promoter consensus sequences affects the binding of transcription factors that maintain endogenous levels of gene expression. This offers an epigenetic mechanism for cell-type-specific control of AhR expression in human tumor cells and points to this receptor as a negative regulator of cell growth and proliferation in ALL. Although in vivo experiments are still underway and results are not yet available, it is tempting to speculate that certain types of ALL tumors could benefit from downregulating AhR expression. This hypothesis, if true, could provide a tumor suppressor activity for the AhR in ALL, a new function further supporting its role in the control of cell cycle progression and cell proliferation in a cell-specific manner and in the absence of xenobiotics. Since promoter hypermethylation is considered to have prognostic value in tumor development, AhR hypermethylation could represent a new biomarker for risk assessment in human cancers such as ALL. The net contribution of lower AhR levels to ALL deserves further investigation.


   Notes
 
{dagger}These authors contributed equally to this work. Back


   Acknowledgments
 
This work has been funded by Grants SAF-2002-00034 and SAF-2005-00130 from the Spanish Ministry of Education and Science and from the Junta de Extremadura (2PR04A060) (to P.F.S.).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Denison,M.S. and Nagy,S.R. (2003) Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Annu. Rev. Pharmacol. Toxicol., 43, 309–334.[CrossRef][ISI][Medline]
  2. Lahvis,G.P., Pyzalski,R.W., Glover,E., Pitot,H.C., McElwee,M.K. and Bradfield,C.A. (2005) The aryl hydrocarbon receptor is required for developmental closure of the ductus venosus in the neonatal mouse. Mol. Pharmacol., 67, 714–720.[Abstract/Free Full Text]
  3. Fernandez-Salguero,P., Pineau,T., Hilbert,D.M., McPhail,T., Lee,S.S., Kimura,S., Nebert,D.W., Rudikoff,S., Ward,J.M. and Gonzalez,F.J. (1995) Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science, 268, 722–726.[Abstract/Free Full Text]
  4. Corchero,J., Martin-Partido,G., Dallas,S.L. and Fernandez-Salguero,P.M. (2004) Liver portal fibrosis in dioxin receptor-null mice that overexpress the latent transforming growth factor-beta-binding protein-1. Int. J. Exp. Pathol., 85, 295–302.[CrossRef][ISI][Medline]
  5. Thurmond,T.S., Staples,J.E., Silverstone,A.E. and Gasiewicz,T.A. (2000) The aryl hydrocarbon receptor has a role in the in vivo maturation of murine bone marrow B lymphocytes and their response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol., 165, 227–236.[CrossRef][ISI][Medline]
  6. Ge,N.L. and Elferink,C.J. (1998) A direct interaction between the aryl hydrocarbon receptor and retinoblastoma protein. Linking dioxin signaling to the cell cycle. J. Biol. Chem., 273, 22708–22713.[Abstract/Free Full Text]
  7. Ito,T., Tsukumo,S., Suzuki,N. et al. (2004) A constitutively active arylhydrocarbon receptor induces growth inhibition of Jurkat T cells through changes in the expression of genes related to apoptosis and cell cycle arrest. J. Biol. Chem., 279, 25204–25210.[Abstract/Free Full Text]
  8. Hayashibara,T., Yamada,Y., Mori,N., Harasawa,H., Sugahara,K., Miyanishi,T., Kamihira,S. and Tomonaga,M. (2003) Possible involvement of aryl hydrocarbon receptor (AhR) in adult T-cell leukemia (ATL) leukemogenesis: constitutive activation of AhR in ATL. Biochem. Biophys. Res. Commun., 300, 128–134.[CrossRef][ISI][Medline]
  9. Koliopanos,A., Kleeff,J., Xiao,Y., Safe,S., Zimmermann,A., Buchler,M.W. and Friess,H. (2002) Increased arylhydrocarbon receptor expression offers a potential therapeutic target for pancreatic cancer. Oncogene, 21, 6059–6070.[CrossRef][ISI][Medline]
  10. Jana,N.R., Sarkar,S., Ishizuka,M., Yonemoto,J., Tohyama,C. and Sone,H. (1999) Cross-talk between 2,3,7,8-tetrachlorodibenzo-p-dioxin and testosterone signal transduction pathways in LNCaP prostate cancer cells. Biochem. Biophys. Res. Commun., 256, 462–468.[CrossRef][ISI][Medline]
  11. Abdelrahim,M., Smith,R.,III and Safe,S. (2003) Aryl hydrocarbon receptor gene silencing with small inhibitory RNA differentially modulates Ah-responsiveness in MCF-7 and HepG2 cancer cells. Mol. Pharmacol., 63, 1373–1381.[Abstract/Free Full Text]
  12. Mancini,D.N., Singh,S.M., Archer,T.K. and Rodenhiser,D.I. (1999) Site-specific DNA methylation in the neurofibromatosis (NF1) promoter interferes with binding of CREB and SP1 transcription factors. Oncogene, 18, 4108–4119.[CrossRef][ISI][Medline]
  13. Santoro,R. and Grummt,I. (2001) Molecular mechanisms mediating methylation-dependent silencing of ribosomal gene transcription.Mol. Cell, 8, 719–725.[CrossRef][ISI][Medline]
  14. Takizawa,T., Nakashima,K., Namihira,M., Ochiai,W., Uemura,A., Yanagisawa,M., Fujita,N., Nakao,M. and Taga,T. (2001) DNA methylation is a critical cell-intrinsic determinant of astrocyte differentiation in the fetal brain. Dev. Cell, 1, 749–758.[CrossRef][ISI][Medline]
  15. Esteller,M. (2002) CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future. Oncogene, 21, 5427–5440.[CrossRef][ISI][Medline]
  16. Chim,C.S., Liang,R. and Kwong,Y.L. (2002) Hypermethylation of gene promoters in hematological neoplasia. Hematol. Oncol., 20, 167–176.[CrossRef][ISI][Medline]
  17. Paz,M.F., Fraga,M.F., Avila,S., Guo,M., Pollan,M., Herman,J.G. and Esteller,M. (2003) A systematic profile of DNA methylation in human cancer cell lines. Cancer Res., 63, 1114–1121.[Abstract/Free Full Text]
  18. Esteller,M., Corn,P.G., Baylin,S.B. and Herman,J.G. (2001) A gene hypermethylation profile of human cancer. Cancer Res., 61, 3225–3229.[Abstract/Free Full Text]
  19. Costello,J.F., Plass,C. and Cavenee,W.K. (2000) Aberrant methylation of genes in low-grade astrocytomas. Brain Tumor Pathol., 17, 49–56.[Medline]
  20. Fraga,M.F., Herranz,M., Espada,J. et al. (2004) A mouse skin multistage carcinogenesis model reflects the aberrant DNA methylation patterns of human tumors. Cancer Res., 64, 5527–5534.[Abstract/Free Full Text]
  21. Ballestar,E., Paz,M.F., Valle,L., Wei,S., Fraga,M.F., Espada,J., Cigudosa,J.C., Huang,T.H. and Esteller,M. (2003) Methyl-CpG binding proteins identify novel sites of epigenetic inactivation in human cancer. EMBO J., 22, 6335–6345.[CrossRef][ISI][Medline]
  22. Eguchi,H., Hayashi,S., Watanabe,J., Gotoh,O. and Kawajiri,K. (1994) Molecular cloning of the human AH receptor gene promoter. Biochem. Biophys. Res. Commun., 203, 615–622.[CrossRef][ISI][Medline]
  23. Kuznetsov,N.V., Andersson,P., Gradin,K., Stein,P., Dieckmann,A., Pettersson,S., Hanberg,A. and Poellinger,L. (2005) The dioxin/aryl hydrocarbon receptor mediates downregulation of osteopontin gene expression in a mouse model of gastric tumourigenesis. Oncogene, 24, 3216–3222.[CrossRef][ISI][Medline]
  24. Nohara,K., Pan,X., Tsukumo,S. et al. (2005) Constitutively active aryl hydrocarbon receptor expressed specifically in T-lineage cells causes thymus involution and suppresses the immunization-induced increase in splenocytes. J. Immunol., 174, 2770–2777.[Abstract/Free Full Text]
  25. Berwick,M., Matullo,G., Song,Y.S., Guarrera,S., Dominguez,G., Orlow,I., Walker,M. and Vineis,P. (2004) Association between aryl hydrocarbon receptor genotype and survival in soft tissue sarcoma. J. Clin. Oncol., 22, 3997–4001.[Abstract/Free Full Text]
  26. Melki,J.R. and Clark,S.J. (2002) DNA methylation changes in leukaemia. Semin. Cancer Biol., 12, 347–357.[CrossRef][ISI][Medline]
  27. Salomon-Nguyen,F., Della-Valle,V., Mauchauffe,M., Busson,Le, Coniat,M., Ghysdael,J., Berger,R. and Bernard,O.A. (2000) The t(1;12)(q21;p13) translocation of human acute myeloblastic leukemia results in a TEL-ARNT fusion. Proc. Natl Acad. Sci. USA, 97, 6757–6762.[Abstract/Free Full Text]
  28. Corn,P.G., Kuerbitz,S.J., van Noesel,M.M., Esteller,M., Compitello,N., Baylin,S.B. and Herman,J.G. (1999) Transcriptional silencing of the p73 gene in acute lymphoblastic leukemia and Burkitt's lymphoma is associated with 5' CpG island methylation. Cancer Res., 59, 3352–3356.[Abstract/Free Full Text]
  29. Agirre,X., Vizmanos,J.L., Calasanz,M.J., Garcia-Delgado,M., Larrayoz,M.J. and Novo,F.J. (2003) Methylation of CpG dinucleotides and/or CCWGG motifs at the promoter of TP53 correlates with decreased gene expression in a subset of acute lymphoblastic leukemia patients. Oncogene, 22, 1070–1072.[CrossRef][ISI][Medline]
  30. Chim,C.S., Wong,A.S. and Kwong,Y.L. (2003) Epigenetic inactivation of INK4/CDK/RB cell cycle pathway in acute leukemias. Ann. Hematol., 82, 738–742.[CrossRef][ISI][Medline]
Received November 8, 2005; revised December 20, 2005; accepted January 8, 2006.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Toxicol SciHome page
S. M. Billiard, J. N. Meyer, D. M. Wassenberg, P. V. Hodson, and R. T. Di Giulio
Nonadditive effects of PAHs on Early Vertebrate Development: mechanisms and implications for risk assessment
Toxicol. Sci., September 1, 2008; 105(1): 5 - 23.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
A. C. Roman, D. A. Benitez, J. M. Carvajal-Gonzalez, and P. M. Fernandez-Salguero
Genome-wide B1 retrotransposon binds the transcription factors dioxin receptor and Slug and regulates gene expression in vivo
PNAS, February 5, 2008; 105(5): 1632 - 1637.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
G. A. Michelotti, D. M. Brinkley, D. P. Morris, M. P. Smith, R. J. Louie, and D. A. Schwinn
Epigenetic regulation of human {alpha}1d-adrenergic receptor gene expression: a role for DNA methylation in Sp1-dependent regulation
FASEB J, July 1, 2007; 21(9): 1979 - 1993.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
27/5/1099    most recent
bgi344v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (7)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Mulero-Navarro, S.
Right arrow Articles by Fernandez-Salguero, P.M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mulero-Navarro, S.
Right arrow Articles by Fernandez-Salguero, P.M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?