Cancer Research Annual Meeting 2010 Jordan
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jiang, R.-C.
Right arrow Articles by Zeng, Y.-X.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jiang, R.-C.
Right arrow Articles by Zeng, Y.-X.
[Cancer Research 66, 693-700, January 15, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

A Functional Variant in the Transcriptional Regulatory Region of Gene LOC344967 Cosegregates with Disease Phenotype in Familial Nasopharyngeal Carcinoma

Ri-Cheng Jiang1,2, Hai-De Qin1,2, Mu-Sheng Zeng1,2, Wei Huang4, Bing-Jian Feng1,2, Feng Zhang1,3, Han-Kui Chen1,2, Wei-Hua Jia1,2, Li-Zhen Chen1,2, Qi-Sheng Feng1,2, Ru-Hua Zhang1,2, Xing-Juan Yu1,2, Mei-Zhen Zheng1,2 and Yi-Xin Zeng1,2

1 State Key Laboratory of Oncology in Southern China; Departments of 2 Experimental Research and 3 Nasopharyngeal Carcinoma, Sun Yat-sen University Cancer Center, Guangzhou, China; and 4 Chinese National Human Genome Center at Shanghai, Shanghai, China

Requests for reprints: Yi-Xin Zeng, Sun Yat-sen University Cancer Center, 651 Dongfeng Road East, Guangzhou 510060, China. Phone: 86-20-8734-3333; Fax: 86-20-8734-3295; E-mail: yxzeng{at}gzsums.edu.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nasopharyngeal carcinoma is a common malignancy in Southeast Asian countries, and genetic background is a well-known component of the complexity underlying its tumorigenic process. We have mapped a nasopharyngeal carcinoma susceptibility locus to chromosome 4p15.1-q12 in a previous linkage study on nasopharyngeal carcinoma pedigrees. In this study provided in this communication, we screened all the genes in this region, with a focus on exons, promoters, and the exon-intron boundary to identify nasopharyngeal carcinoma–associated mutations or functional variants. Importantly, we found a novel gene (LOC344967) with a single nucleotide polymorphism –32G/A in the promoter region. This gene is a member of the acyl CoA thioesterase family that plays an important role in fatty acid metabolism and is involved in the progression of various types of tumors. The –32A variant was found cosegregated with the disease phenotype in the nasopharyngeal carcinoma pedigrees that we previously used for the linkage study. Moreover, this –32A variant creates an activator protein (AP-1)–binding site in the transcriptional regulatory region of LOC344967, which significantly enhanced the binding of AP-1 to the promoter region and the transcription activity of the promoter in vivo. Furthermore, the expression of LOC344967 was significantly up-regulated at both mRNA and protein levels in nasopharyngeal carcinoma cells sharing the –32G/A genotype compared with nasopharyngeal carcinoma cells with the –32G/G genotype. Collectively, these results provide evidence that the –32A variant is a functional sequence change and may be related to nasopharyngeal carcinoma susceptibility in the families studied. (Cancer Res 2006; 66(2): 693-700)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nasopharyngeal carcinoma is a human malignancy derived from epithelia cells and is rare in most parts of the world but is common in Southern China, Southeast Asian, and in some North African countries (1). Nasopharyngeal carcinoma represents a superb model of gene-environment-virus interaction in the pathogenesis of cancer. Consumption of salted fish has been revealed as a risk factor for nasopharyngeal carcinoma. EBV has been regarded as related to and, perhaps, a probable cause of nasopharyngeal carcinoma. A vast amount of evidence strongly suggests that genetic factors provide a background susceptibility on which EBV and environmental factors act on and eventually result in nasopharyngeal carcinoma development (2–5).

However, how and what genes contribute to nasopharyngeal carcinoma–related genetic susceptibility still remains to be fully clarified. In 1975, Simons et al. first reported the association between the HLA locus and nasopharyngeal carcinoma risk (6), and a number of subsequent studies have confirmed this finding (7–10). Moreover, Lu et al. carried out an affected sib pair linkage analysis, and their results suggested a linkage between the HLA region and the nasopharyngeal carcinoma phenotype (11). Very recently, Lu et al. further localized the susceptibility locus to a 132-kb segment containing the HLA-A locus, but the exact gene has yet to be identified (12–14). Besides the HLA locus or hyplotypes, a susceptibility locus for familial nasopharyngeal carcinoma has been mapped to chromosome 3p21 (15), and polymorphism of the genes of T-cell receptors (16), glutathione S-transferase MI (17), cytochrome P450 2E1 (18), polymeric immunoglobulin receptor (19), stress protein HSP70-2 (20), DNA repair enzymes XRCC1 and hOGG1 (21), and certain other genes (22–25) have also been reported to be associated with risk of nasopharyngeal carcinoma. A most likely scenario would be that a number of different genes are involved in different signal pathways or cellular function activities, thus, together would contribute to define a genetic specificity for nasopharyngeal carcinoma; such is in support for a multifactorial mode of inheritance (26).

Familial clustering of nasopharyngeal carcinoma has been well documented (27, 28), and in our previous study on 2,252 nasopharyngeal carcinoma probands, we found that nasopharyngeal carcinoma tended to aggregate in Cantonese families more than in other dialect-speaking populations in Guangdong Province of Southern China (29). By genome-wide linkage analysis with microsatellite markers on 32 Cantonese nasopharyngeal carcinoma pedigrees, we mapped the nasopharyngeal carcinoma susceptibility locus to chromosome 4p15.1-q12 (30). Subsequently, by adding more microsatellite and single nucleotide polymorphism (SNP) markers, we further narrowed down the susceptibility region from 14.21 to 8.29 cM at 4p11-p14 (31). As a result of the progress of the Human Genome Project, we were able to acquire the genomic information of this region; the list of the genes in the region has been published very recently in Nature (32). In an effort to identify the nasopharyngeal carcinoma–associated mutations or functional variants, we began to screen all the genes in this region with a focus on exons, promoter, and the exon-intron boundary. In this communication, we report our finding of a novel gene, LOC344967, with a SNP –32A/G in the promoter region cosegregated with the disease haplotype in three largest nasopharyngeal carcinoma pedigrees we previously used for the genome-wide linkage study. Moreover, this SNP –32A results in an activator protein (AP-1)–binding site that significantly enhances the binding of AP-1 to the promoter region in vitro and increases transcription activity in vivo. Thus, our results identify a functional sequence variant that may contribute substantially to susceptibility to familial nasopharyngeal carcinoma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Nasopharyngeal carcinoma pedigrees. The 32 nasopharyngeal carcinoma families were recruited from the Pearl River area in Guangdong Province, China, and they all speak the Cantonese dialect. These families were used for our previous genome-wide linkage study. Detailed descriptions of their clinicopathologic characteristics were reported by Feng et al. (30).

SNP analyses. Screening for SNP was first done on six individuals selected from the two largest pedigrees, 31 and 34; such contributed to the linkage results in a previous study (30). Subsequently, the candidate SNPs were expanded to all the 32 pedigrees for further confirmation. The cases from pedigree 31 include 31-35 and 31-44; 31-15 is a normal control that does not share the disease haplotype. The cases from pedigree 34 include 34-5 and 34-9; the control is 34-11 (30). We focused on the interval spanning from D4S2950 to D4S2916 (37-55 Mb), ~17Mbp in length, harboring 78 confirmed genes, 14 predicted genes, and seven pseudo genes according to the Human Genome Database of National Center for Biotechnology Information (NCBI; Build 35.1). Primers were designed using Primer 3.0 (http://frodo.wi.mit.edu/), and the expected products are ~600 bp in length, with a 100-bp overlap with the adjacent fragment. The exonic and flanking intronic sequence and the promoter region of the genes were covered. We presumed that 2 to 3 kb upstream of the first exon of the genes to be the promoter region; such would be analyzed by the promoter prediction programs (e.g., FirstEF, Promoter2.0, NNPP2.2, etc.; ref. 33). Inappropriate primers would be redesigned whenever failure appeared in the PCR or sequencing step. Sequencing reactions were done on an ABI377 or ABI3730 sequencer (PE Applied Biosystems, Foster City, CA). We base-called sequences, assembled contigs, and detected SNPs using a Polyphred package (Polyphred, Phred/Phrap/Consed, http://droog.gs.washington.edu/); all traces were visually inspected by at least two observers.

Cell culture. Human embryonal kidney HEK293 cells were cultured in RPMI 1640 (Life Technologies, Carlsbad, CA) supplemented with 10% FCS. The human nasopharyngeal carcinoma cell lines CNE1, CNE2, and C666 were cultured in RPMI 1640 supplemented with 10% FCS. NP69 cells, a SV40 T antigen-immortalized nonmalignant nasopharyngeal epithelial cell line, were cultured in keratinocyte-SFM (Life Technologies) with L-glutamine, epidermal growth factor and bovine pituitary extract. NP69 and C666 cells were obtained from Prof. S.W. Tsao (Department of Anatomy, University of Hong Kong, Hong Kong, China). All cells were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37°C.

Plasmid constructions. LOC344967 promoter regions (–1,008 bp from ATG, based upon an open reading frame analysis of AK125299, on the Japanese NEDO Human cDNA Sequencing Project database) containing –32G or –32A alleles were cloned into the KpnI/NcoI sites of pGL3-Basic vectors (Promega, Madison, WI) upstream from the luciferase gene. All the recombinants were confirmed by sequencing the plasmid DNA.

Preparation of nuclear extracts. Nuclear extracts from HEK293 cells were prepared with the NE-PER kit (Pierce, Rockford, IL) according to the manufacturer's instruction. Protein concentrations were determined by a bicinchoninic acid (BCA) assay (Pierce), and the nuclear extracts were stored at –80°C.

Electrophoretic mobility shift assay. Single-stranded, complementary oligonucleotide sequences corresponding to the 21-bp fragments with the two different alleles (G and A) were synthesized (Shanghai Bioasia Biotechnology Co., Ltd., Shanghai, China) and annealed to form double-stranded probes. Unlabeled consensus AP-1 motif double-stranded oligonucleotides (1.75 pmol/µL) were purchased from Promega. AP-1 antibodies specific for individual components of AP-1 (c-fos and c-Jun) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Radioactive probes were generated by labeling a single-stranded oligonucleotide with a T4 polynucleotide kinase and [{gamma}-32P]ATP (5,000 Ci/mmol at 10 mCi/mL, from Beijing FuRui Co., Beijing, China) and subsequently annealing the complementary oligonucleotide and purification through a Sephadex G25 Quickspin column (Roche Diagnostics Ltd., Indianapolis, IN). Binding reactions were done for 10 minutes at room temperature using 5 to 10 µg of HEK293 cell nuclear extract and 30,000 counts/min radiolabeled probe in a binding buffer containing 10 mmol/L Tris-HCl (pH 7.5), 50 mmol/L NaCl, 1 mmol/L MgCl2, 0.5 mmol/L EDTA, 4% glycerol, 0.5 mmol/L DTT, and 0.1 mg poly(deoxyinosinic-deoxycytidylic acid). In competition experiments, a cold competitor (50x and 200x molar excess) was incubated with the nuclear extract for 10 minutes before the addition of the labeled probe. In supershift experiments, 1.0 µg antibody was incubated with the nuclear extract for 3 hours at 4°C after probe addition. Protein/DNA complexes were separated in 6% nondenaturing polyacrylamide gels. The gels were dried and exposed to X-ray films at –80°C overnight. The sequences of the oligonucleotide probes are as follows: AP-1-like oligo (allele A), 5'-GCTAATGTGACCCGCAATGTC-3'; oligonucleotide probe (allele G), 5'-GCTAATGTGGCCCGCAATGTC-3'; AP-1 consensus oligonucleotide, 5'-CGCTTGATGAGTCAGCCGGAA-3' and its reverse complements.

Chromatin immunoprecipitation assay. Subconfluent NP69 cells carrying the –32G/A grown in keratinocyte-SFM medium or CNE2 cells carrying –32G/G genotypes or grown in RPMI 1640 were cross-linked by treatment with 1% formaldehyde for 15 minutes. The chromatin immunoprecipitation assay was carried out as reported earlier (34). Briefly, equal aliquots of isolated chromatin were subjected to immunoprecipitation with a rabbit anti-AP-1 (anti-c-Jun and anti-c-Fos) antibodies, rabbit serum or PBS control. The DNA associated with specific immunoprecipitates or with negative control serum or PBS was isolated and used as a template for PCR to amplify the promoter sequences of the LOC344967 containing the AP-1-like element. The primers used were 5'-primer, 5'-ACACAGCGATCCCCGTTC-3' and 3'-primer, 5'-CTGGCCCAGACATCAAGAC-3'; and the expected product size is 164 bp. As a positive control, the MSH2 promoter, which contains the functional AP-1 element, was amplified from the same template using the following primers: 5'-primer, 5'-TGCCACAAACGCATATCTTC-3'; 3'-primer, 5'-CGACTGCCTCCCTTTTTCT-3'; and the expected product size is 300 bp (35).

Transient transfection and luciferase assay. For the luciferase assay, HEK293 cells were seeded in six-well microplates 1 day before transfection and grown to 70 to 80% confluence. Either one of the LOC344967 promoter-luciferase constructs (1.6 µg) was cotransfected with 0.4 µg of the pEFRL Renilla luciferase as an internal control for transfection efficiency by 2.0 µL LipofectAMINE 2000. To assess background level of luciferase activity, pGL3-Basic vector without enhancer and/or promoter regions of the LOC344967 gene was cotransfected with the pEFRL Renilla luciferase vector. Cells were harvested 24 hours after transfection, and luciferase activities were determined by the Dual-Luciferase Reporter Assay System (Promega) in a Monolight 3010 luminometer. Firefly luciferase activities were normalized by Renilla luciferase activities. All transfection experiments were done a minimum of three times.

DNA PCR and mRNA reverse transcription-PCR. DNA and total RNA were isolated from NP69, C666, CNE1, and CNE2 cells. The first-strand cDNA synthesis was done with the SuperScript First-Strand Synthesis System (Invitrogen, San Diego, CA). DNA was amplified with primer pairs (5'-TCCATCTGAATTGAAGGGATT-3' and 5'-GGCCTTATTGGTCAGCTTTT-3') to identify the allele at –32 bp. Reverse transcription-PCR (RT-PCR) was used to analyze LOC344967 gene transcription level with the primer pairs (5'-CTCTGGAACTCGAAGGATCA-3' and 5'-GCACCTGTGAGGATGTTTTC-3'). The 364-bp RT-PCR product was sequenced for confirmation.

Quantification of LOC344967 expression using real-time RT-PCR in cell lines with –32G/A or G/G genotypes. Quantitative real-time RT-PCR analysis was done using the Rotor-Gene3000 and SYBRGreen systems (Corbett Research, Sydney, Australia) in accordance with manufacturer's instructions. Data were normalized by the level of ß-actin expression in each of the individual samples. We designed primers using the primer design software Primer Express 2.0 (PE Applied Biosystems). To ensure that the primer amplified the desired cDNA segment, the forward and reverse primers were located on different exons and were checked in the BLAST program in NCBI and Celera database. There was only one PCR product from the pair of primers as seen by electrophoresis, and the product was sequenced to confirm that the correct gene was amplified. Primers for LOC344967 were used as the following: 5'-GCCGCAGAAAGGGTCTCA-3' (sense) and 5'-GATGGCGTTCGGCGTCTT-3' (antisense). Primers for ß-actin were used as the following: 5'-CCTGTACGCCAACACAGTGC-3' (sense) and 5'-ATACTCCTGCTTGCTGATCC-3' (antisense).

Generation of antigen peptides and antibodies and Western blotting detection. According to mRNA sequence and amino acid sequence of AK125299 (gi: 34531348; NEDO human cDNA sequencing project), the antigen peptide (amino acids 102-118, N'-CYFRQEQEEEGQKRYKT) was designed and synthesized by Proteintech Group. Inc. (Chicago, IL). Cysteine was added for keyhole limpet hemocyanin (KLH) conjugation purpose in the peptide. Polyclonal antiserum (S1107) to hLOC344967 was obtained after immunization of rabbits with the KLH-coupled internal peptide of hLOC344967 (Proteintech Group). Protein concentrations of cell lysate from NP69, C666, CNE1, and CNE2 were determined by BCA assay (Pierce), and 15 µg total protein from each cell line were subjected to 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. We blocked the membrane with 5% skimmed milk in TBST [20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.05% Tween 20] and probed it with a polyclonal antiserum to LOC344967 at 1:200 dilution. After rinsing in TBST (3 x 10 minutes), the membrane was incubated with an appropriate horseradish peroxidase–linked secondary antibody for 1.5 hours at room temperature. After a final rinsing with TBST (3 x 10 minutes) to remove unbound antibody, specific proteins were visualized by the enhanced chemiluminescence method (Cell Signaling Technology, Beverly, MA) and exposed to Kodak X-ray films.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
–32A variant located in the promoter region of LOC344967 cosegregated with nasopharyngeal carcinoma disease haplotype. SNP discovery strategy was employed to screen mutations in the susceptibility locus we previously mapped to chromosome 4p15.1-q12. We examined most of the genes in the region of six individuals (four cases and two controls), who were chosen from the two largest nasopharyngeal carcinoma pedigrees 31 and 34, which showed strong linkage to nasopharyngeal carcinoma in our previous genome-wide linkage study (30). We screened totally 78 confirmed genes and six predicted genes with a focus on exonic and flanking intronic sequence and the promoter region of the genes as well. We identified about 600 SNPs in this region, and nearly one third of them were not listed in the dbSNP or Celera SNP database (data not shown). This allowed us to find mutations cosegregated with the disease phenotype. One polymorphism (reference ID, rs17585937; –32A) in the transcriptional regulatory region of the gene LOC344967 was found to cosegregate with the disease phenotype in the six individuals. LOC344967 is located at chromosome 4p14 (NCBI Human Genome Project database, Build 35.1) and shares about 89% homology with a cytosolic acyl CoA thioester hydrolase (EC 3.1.2.2) and is most likely a novel member of the thioesterase superfamily (NEDO Human cDNA Sequencing Project database). As shown in Fig. 1, among the six individuals from pedigree 34 (34-5, affected; 34-9, affected; 34-11, nonaffected) and pedigree 31 (31-35. affected; 31-44, affected; 31-15, nonaffected), four cases share the allele A, as a heterozygous G/A genotype. Besides this –32A variant, we also identified some other SNP cosegregated with disease phenotype in pedigree 31 or 34 but not in both of them. For example, there is a cSNP 78T->A, resulting in amino acid change CAT26CAA (His26Gln), in cytochrome oxidase VIIb2 (COX7B2) gene, which was found in the nasopharyngeal carcinoma cases of pedigree 31 (36).


Figure 1
View larger version (38K):
[in this window]
[in a new window]
  		Figure 1. Traces of six individuals in pedigree 34 and 31. A, traces of 34-5 (affected), 34-9 (affected), and 34-11 (nonaffected). B, traces of 31-35 (affected), 31-44 (affected), and 31-15 (nonaffected). The sequences were further confirmed by sequencing from the reverse direction.

 
Subsequently, we expanded the SNP detection in LOC344967 to all the familial members of the 32 pedigrees studied in our previous linkage analysis. As shown in Fig. 2, each of the individuals sharing the potential nasopharyngeal carcinoma susceptibility haplotype in pedigrees 31, 34, and 62 were found to carry the SNP –32A in LOC344967. These families are the three largest pedigrees in terms of case numbers; in addition, they were the main contributors to the total logarithm of odds (LOD for linkage, and 3.0 was usually considered as a threshold for significance) score of 3.67 in our previous linkage study. LOD scores of pedigrees 31, 34, and 62 were 1.646271, 1.618488, and 0.700149, respectively, and the probabilities of linkage were 0.9659, 0.9637, and 0.7622, respectively (30). These results indicate that –32A is genetically linked to the nasopharyngeal carcinoma phenotype in some of the high-risk nasopharyngeal carcinoma pedigrees. In the remaining 29 pedigrees, about one third of the cases share the –32A variant, and because of the small size of the pedigrees and the ambiguity in linkage, we did not expect all the cases to share the variant as in the three largest pedigrees.


Figure 2
View larger version (18K):
[in this window]
[in a new window]
  		Figure 2. The potential etiological allele segregated with susceptibility haplotype or nasopharyngeal carcinoma phenotype. The three nasopharyngeal carcinoma pedigrees used in a previous linkage study are shown, and their LOD scores are indicated. The number of the first row below the circles or squares denotes the member ID in the pedigrees. For the second row, the number 0, 1, or 2 indicates the genotype unknown, allele G, or allele A, respectively.

 
Transcription factor AP-1 binding to the –21/–41 bp region with allele A of the LOC344967 promoter. We then investigated the potential role of the SNP –32A by examining the promoter region with the MatInspector V2.2 program of the TRANSFAC database. We found that the variation –32A in the primary genomic sequence resulted in a potential binding site for transcriptional factor AP-1 in the region of –26/–36 bp of LOC344967 5'-untranslated region (Fig. 3A). We synthesized two oligonucleotides containing the –21 to –41 region with the –32A or –32G allele and carried out a gel shift assay. As shown in Fig. 3B, the 32P-labeled oligonucleotide (allele A) bound specifically to nuclear extracts from HEK293 cells and formed a shifted complex at the same position as the AP-1 consensus oligonucleotide. But the oligonucleotide with allele G did not form such a complex (Fig. 3B, lane 5). This was confirmed by supershift with antibodies to AP-1 and a complete inhibition by 200-fold unlabeled consensus AP-1 oligonucleotide and the allele A–containing oligonucleotide (Fig. 3B).


Figure 3
View larger version (37K):
[in this window]
[in a new window]
  		Figure 3. Transcription factor binding to the putative AP1-like motif in the –41/–21 region of the LOC344967 promoter. A, schematic illustration of the –54/3 region of the LOC344967 promoter indicating positions of the putative AP1 transcription factor binding motif (underlined). The sequences of wild-type (G) and mutant (A) (in red) oligonucleotide probes used for gel shift analysis is indicated by the brackets. Nucleotide numbering is according to AK125299 sequences (gi: 34531348. NEDO Human cDNA Sequencing Project). B, Gel shift, supershift and competition assays with HEK293 cell nuclear extract and oligonucleotides with different allelic variants of the LOC344967 promoter. Binding was present with the AP1-like oligonucleotide containing allele A (lane 4, 7 and 9) and AP1 consensus oligonucleotide (lane 3), but was not observed with oligonucleotide containing allele G (lane 5) when HEK293 cell extract was present. Binding to oligonucleotide with allele A resulted in a complex that was completely competed (cold oligos) by 200-fold molar excess of unlabeled AP1-like oligonucleotide containing allele A (lane 8) and AP1 consensus oligonucleotide (lane 6), but was not competed by the same amount of unlabeled oligonucleotide containing allele G (lane 9). AP1-like oligonucleotide containing allele A supershift was present with antibodies against c-Jun (lane 11) and c-Fos (lane 13). AP1 consensus oligonucleotide supershift was present with antibodies against c-Jun (lane 10) and c-Fos (lane 12).

 
To test whether the binding also occur in vivo, we carried out a chromatin immunoprecipitation assay on one nasopharyngeal carcinoma cell line CNE2 with –32G/G genotype and one immortalized nonmalignant nasopharyngeal epithelial cell line NP69 with –32G/A genotype. As shown in Fig. 4, a clear PCR amplification product of the LOC344967 promoter was observed in chromatin immunoprecipitation carried out with anti-AP-1 antibody only in NP69 cells but not in CNE2 cells (Fig. 4, bottom, lanes 5 and 9). For the positive control, MSH2 promoter (35), a clear band was observed in both CNE2 and NP69 cells (Fig. 4, top, lanes 5 and 9) but not for the negative controls, rabbit serum, or PBS (Fig. 4, top and bottom, lanes 3, 4 and 7, 8). Collectively, these data suggest that there was an AP-1-binding motif at the –21/–41 bp region because of the substitution –32G/A, and transcription factor AP-1 was able to specifically bind to this region in vitro and in vivo.


Figure 4
View larger version (28K):
[in this window]
[in a new window]
  		Figure 4. ChIP analysis of AP1 binding to the putative AP1-like motif of the LOC344967 promoter. CNE2 and NP69 cells were cultured and chromatin lysates of these two cell lines were prepared as described under "Materials and Methods." Equal aliquots of chromatin lysates were subjected to immunoprecipitation with antibodies against AP1 (anti-c-Jun/anti-c-Fos mixture) (lane 5 and 9), rabbit serum control (lane 4 and 8) or blank (lane 3 and 7) with PBS control. The DNA associated with immunoprecipitates was isolated and used as templates to PCR-amplify the positive control gene MSH2 promoter region containing a known functional AP1 binding site (upper panel) or the LOC344967 promoter containing the putative AP1-like motif (lower panel). The PCR products were resolved by agarose gel electrophoresis and stained with ethidium bromide.

 
–32A variant located in the promoter region of LOC344967 generated one functional cis-regulatory element and was associated with greater promoter activity. To further determine the effects of the –32A variant on AP-1-induced LOC344967 transcriptional activation, transient transfection assays with the luciferase reporters driven by the LOC344967 promoter segment containing either allele A or G were done in HEK293 cells. We cloned both the –1,008-bp region containing –32A or G of the LOC344967 promoter into the pGL3 vector upstream of the luciferase reporter. Those constructs were transfected into the HEK293 cells, and luciferase activity was measured 24 hours after transfection. As we expected, the luciferase activity of the construct with allele A was 1.83 times greater than that with allele G (P < 0.0001; Fig. 5). These results suggest that the –32A variant is functionally significant.


Figure 5
View larger version (9K):
[in this window]
[in a new window]
  		Figure 5. The substitution –32G->A enhanced transcription activity of LOC344967 promoter in vivo. The constructs of LOC344967 promoter reporters are shown in the left side of the figure, which include both the –1008 bp promoter region with allele A and G as indicated. Luciferase activity (mean ± s.d., n = 3) is measured 48 hours after transient transfection of the plasmid constructs into HEK293 cells which are rich in AP1 transcription factors. The results are presented in relative luciferase activity (normalized by pEFRL Renilla luciferase activities). The activity of the control vector without insert (pGL3-Basic) was used to measure the background level. Relative luciferase activity of the reporter construct with allele A was 1.83 times greater than that with allele G (P < 0.0001).

 
LOC344967 is widely expressed in multiple tissues, and its mRNA and protein expression levels were associated with the –32A variant. To investigate the tissue distribution of LOC344967, we detected LOC344967 mRNA expression in multiple tissues and in the nasopharyngeal carcinoma cell lines by RT-PCR analysis. LOC344967 transcript was detected in all of the tissues, including liver, lung, kidney, brain, and intestine, and in several nasopharyngeal carcinoma cell lines (data not shown). This wide expression pattern may indicate an important role for this gene. Next, we sought to determine if there is a relationship between the –32 variant and the mRNA expression levels. For this purpose, we checked and found two nasopharyngeal carcinoma cell lines CNE1 and CNE2 carrying the –32G/G and two cell lines NP69 and C666 carrying the –32G/A genotypes (Fig. 6A). These cell lines are very popularly used in the field of nasopharyngeal carcinoma research, and three of them are derived from nasopharyngeal carcinoma cells, and only NP69 is a SV40 T antigen–immortalized nasopharyngeal epithelial cell line. Quantitative real-time RT-PCR analysis was used to detect the expression levels of LOC344967. We found a significantly greater expression level in the –32 G/A heterozygous cells NP69 and C666 than in the homozygous –32G/G cells CNE1 and CNE2 (Fig. 6B). Furthermore, we designed a peptide to make polyclonal antibodies, based on analysis of the putative amino acid sequence of LOC344967. The molecular weight of the encoded protein of LOC344967 is ~28 kDa (252 amino acids); a protein band of similar size was detected by Western blotting with the antibody. As shown in Fig. 6C, the protein levels in NP69 and C666 cells were notably higher than in CNE1 and CNE2 cells.


Figure 6
View larger version (26K):
[in this window]
[in a new window]
  		Figure 6. Endogenous LOC344967 mRNA expression level is associated with the –32G/A variants in NPC cell lines. A, the sequence of LOC344967 was determined and two NPC cell lines (CNE1 and CNE2) are –32G/G homozygous and one NPC cell line (C666) and one immortalized nasopharyneal epithelial cell line (NP69) are –32G/A heterozygous in genotype. B, quantitative real-time RT-PCR method was used to measure the expression level of LOC344967 and the results were shown as relative expression units of the gene in 5 µg of total RNA normalized by the internal control ß-actin. The experiment was repeated and a similar result was obtained. C, LOC344967 protein expression was detected in the cell lines. An antigen peptide was designed to make polyclonal antibody as described in Materials and Methods. Equal amounts of total protein (15 µg) from the four cell lines were separated in 12% SDS-PAGE and immunoblotted with the antibody and visualized with the ECL method. The putative molecular weight is 28 kd. ß-actin protein was used as the control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Nasopharyngeal carcinoma is a complex disease with a proven genetic component. This disease is regarded by many to be an excellent and has been regarded as a good model for studying gene-environment-virus mechanistic interactions. However, the precise genetic component in this disease has remained largely elusive. In the elucidation of this genetic component, we have considered two alternative hypotheses: (a) Only one copy of the susceptibility allele has survived in the high-risk population, with long-conserved haplotypes being observed, such has been shown in previous studies of other complex diseases (37–39). (b) The common disease/common variant hypothesis, suggesting a founder effect that is common to the high-risk population and that complex traits have underlying genetics variants with high frequency. Such mutations may have occurred some 100,000 years ago, undergoing little or no selection during population expansion (40). Whether the rare variants or the common variants account for the genetic complexity has been discussed extensively. The variant –32A, in gene LOC344967, as identified in this study, matched well with the nasopharyngeal carcinoma disease phenotype (Figs. 1 and 2). This suggests that the variant might be a high risk factor in high-risk familial nasopharyngeal carcinoma. We have previously carried out a complex segregation analysis based on 1,903 Cantonese pedigrees, and our results provided evidence for a multifactorial mode of inheritance (26). We may assume that –32A was one of such risk factors, and this factor, together with sequence variants in other genes, infection of certain subtypes of EBV, and exposure to environmental carcinogens, could eventually result in nasopharyngeal carcinoma development (3). Certainly, at this stage, we could not rule out the possibility of certain rare mutations with a much higher risk existing in the susceptibility region. One rare variant CAT26CAA (His26Gln) was identified in COX7B2 gene in the pedigree 31 but not in other pedigrees nor sporadic nasopharyngeal carcinoma, and its real relationship with nasopharyngeal carcinoma needs to be further determined (36).

In addition to the genetic evidence, supporting a role of the variant –32A of LOC344967 in relationship with familial nasopharyngeal carcinoma, we have also provided functional evidence. Our in vitro gel shift, supershift, and competition assay and in vivo chromatin immunoprecipitation assay have clearly showed that the AP-1-binding site was formed from the variant compared with the –32G sequence (Figs. 3 and 4). Furthermore, our construct containing the –1,008-bp promoter region with the –32A allele and a luciferase reporter produced significantly greater luciferase activity than the construct with the –32G allele (Fig. 5). These results indicate an enhanced transcription of LOC344967 with the –32A allele through the newly formed AP-1-binding site. The rationale to link such a mechanism to nasopharyngeal carcinoma development may be explained, at least in part, by the oncogenic potential of the EBV-encoded LMP1 protein. LMP1 has been reported to be an important protein, tightly linked to apoptosis prevention and malignant transformation, through activating transcription factors nuclear factor-{kappa}B (NF-{kappa}B) and AP-1 (41–43). In addition, a mutant at amino acids 204, 206, 208, and 384 has been reported to significantly inhibit LMP1-stimulated NF-{kappa}B and AP-1 transcriptional activity (41). Furthermore, a SNP in matrix metalloproteinase-1 promoter creates an Ets-binding site for LMP1 resulting in enhanced susceptibility to nasopharyngeal carcinoma (44). On the other hand, the most abundant polyadenylated viral transcripts of EBV, the so-called complementary-strand transcripts, contain a functional AP-1 regulatory element in its promoter region (45). Thus, it seems that AP-1 plays a central role and is substantially involved in EBV-induced cell transformation.

An important question is how an up-regulated LOC344967 might contribute to nasopharyngeal carcinoma development. By sequence alignment, LOC344967 shares similarity to a cytosolic acyl CoA thioester hydrolase (EC 3.1.2.2, located in chromosome 1p36.31-p36.11; refs. 46, 47), which belongs to a family containing a wide variety of enzymes, principally the thioesterases. This family includes various cytosolic long-chain acyl-CoA thioester hydrolases. Long-chain acyl-CoA hydrolases hydrolyze palmitoyl-CoA to CoA and palmitate and catalyze the hydrolysis of other long-chain fatty acyl-CoA thioesters. These enzymes can be present in a membrane-bound form in subcellular organelles and in a soluble form inside mitochondria and cytosol (46). An important function of these enzymes is determining the chain length of the synthesized fatty acids. Previous studies have established a link between cancer development and fatty acid synthesis (48). For example, fatty acid synthase (FAS) has been reported to be up-regulated in prostate cancer cells compared with normal prostate epithelial cells and has been implicated in the progression of various types of tumors (49–52). Orlistat, an inhibitor of the thioesterase activity of FAS, can block cellular fatty acid synthesis; this results in an inhibition of prostate tumor cell proliferation and an induction of apoptosis in prostate tumor cells (53). CoA thioesterase activity has also been reported to be induced by hypolipidemic peroxisome–proliferating agents leading to an activation of retinoid X receptors and an initiation of transcription of downstream molecules (46). Furthermore, EBV-encoded BZLF1 can physically and functionally interact with the retinoic acid receptors (54).

With regard to LOC344967, although its biological function remains to be determined, we have successfully detected its expression in a variety of normal human tissues, nasopharyngeal carcinoma biopsy specimens, and nasopharyngeal carcinoma cell lines by the RT-PCR method (data not shown). The widely expressed pattern of LOC344967 suggests a potentially important function of this gene. Considering the ubiquitous existence of the AP-1 transcription factor, we suggest that the –32A variant could result in changes in the expression level of LOC344967. To test this hypothesis, we selected four cell lines with two sharing the –32G/G and two sharing the –32G/A genotypes. Each cell line was derived from nasopharyngeal epithelial cells: three are malignant epithelial cell lines and one is immortalized epithelial cell line. LOC344967 expression levels were significantly up-regulated at both the mRNA and protein levels in the two cell lines containing –32G/A genotype. It is conceivable, therefore, that the up-regulation of LOC344967 could result in higher enzyme activity in the epithelial cells, substantially altering fatty acid metabolism and consequently promoting neoplastic cell transformation. Undoubtedly, future studies will more firmly establish the relationship between fatty acid metabolism and epithelial cell–derived cancer development, such studies will shed light on the extremely complicated mechanism of cancer development, which, most probably, will involve multiple genes related to various cellular activities, including, potentially, fatty acid metabolism.


   Acknowledgments
 
Grant support: Ministry of Science and Technology of China and the Natural Science Foundation of Guangdong Province, China 863 and 973 projects.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Takuji Shirasawa and Takahiko Shimizu (Tokyo Metropolitan Institute of Gerontology, Japan) for assistance in antigen peptide design, Prof. Hong-Di Jia (Department of Biochemistry, Beijing University Medical School) for technical helps with the chromatin immunoprecipitation assay, and all patients and their families for participation in this study.


   Footnotes
 
Note: R-C. Jiang, H-D. Qin, and M-S. Zeng contributed equally to this work.

Received 6/21/05. Revised 9/25/05. Accepted 10/31/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Yu MC, Yuan JM. Epidemiology of nasopharyngeal carcinoma. Semin Cancer Biol 2002;12:421–9.[CrossRef][Medline]
  2. Zeng Y, Zhong JM, de-The G, Wu SH, Hou YT, Miao XQ. Enhancement of spontaneous VCA and EA induction in B95-8 cells and EA induction in Raji cells treated with human leukocyte interferon. Intervirology 1982;18:33–7.[Medline]
  3. Zeng YX, Jia WH. Familial nasopharyngeal carcinoma. Semin Cancer Biol 2002;12:443–50.[CrossRef][Medline]
  4. Young LS, Rickinso AB. Epstein-Barr virus: 40 years on. Nat Rev Cancer 2004;4:757–68.[CrossRef][Medline]
  5. Yang XR, Diehl S, Pfeiffer R, et al. Chinese and American Genetic Epidemiology of NPC Study Team. Evaluation of risk factors for nasopharyngeal carcinoma in high-risk nasopharyngeal carcinoma families in Taiwan. Cancer Epidemiol Biomarkers Prev 2005;14:900–5.[Abstract/Free Full Text]
  6. Simons MJ, Wee GB, Chan SH, Shanmugaratnam K, Day NE, de-The G. Immunogenetic aspects of nasopharyngeal carcinoma (NPC) III. HL-a type as a genetic marker of NPC predisposition to test the hypothesis that Epstein-Barr virus is an etiological factor in NPC. IARC Sci Publ 1975;(11 Pt 2):249–58.
  7. Hildesheim A, Apple RJ, Chen CJ, et al. Association of HLA class I and II alleles and extended haplotypes with nasopharyngeal carcinoma in Taiwan. J Natl Cancer Inst 2002;94:1780–9.[Abstract/Free Full Text]
  8. Goldsmith DB, West TM, Morton R. HLA associations with nasopharyngeal carcinoma in Southern Chinese: a meta-analysis. Clin Otolaryngol Allied Sci 2002;27:61–7.
  9. Pimtanothai N, Charoenwongse P, Mutirangura A, Hurley CK. Distribution of HLA-B alleles in nasopharyngeal carcinoma patients and normal controls in Thailand. Tissue Antigens 2002;59:223–5.[CrossRef][Medline]
  10. Hirankarn N, Kimkong I, Mutirangura A. HLA-E polymorphism in patients with nasopharyngeal carcinoma. Tissue Antigens 2004;64:588–92.[CrossRef][Medline]
  11. Lu SJ, Day NE, Degos L, et al. Linkage of a nasopharyngeal carcinoma susceptibility locus to the HLA region. Nature 1990;346:470–1.[CrossRef][Medline]
  12. Lu CC, Chen JC, Jin YT, Yang HB, Chan SH, Tsai ST. Genetic susceptibility to nasopharyngeal carcinoma within the HLA-A locus in Taiwanese. Int J Cancer 2003;103:745–51.[CrossRef][Medline]
  13. Lu CC, Chen JC, Tsai ST, et al. Nasopharyngeal carcinoma-susceptibility locus is localized to a 132 kb segment containing HLA-A using high-resolution microsatellite mapping. Int J Cancer 2005;115:742–6.[CrossRef][Medline]
  14. Hu SP, Day NE, Li DR, et al. Further evidence for an HLA-related recessive mutation in nasopharyngeal carcinoma among the Chinese. Br J Cancer 2005;92:967–70.[CrossRef][Medline]
  15. Xiong W, Zeng ZY, Xia JH, et al. A susceptibility locus at chromosome 3p21 linked to familial nasopharyngeal carcinoma. Cancer Res 2004;64:972–4.
  16. Chen Y, Chan SH. Polymorphism of T-cell receptor genes in nasopharyngeal carcinoma. Int J Cancer 1994;56:830–3.[Medline]
  17. Nazar-Stewart V, Vaughan TL, Burt RD, Chen C, Berwick M, Swanson GM. Glutathione S-transferase M1 and susceptibility to nasopharyngeal carcinoma. Cancer Epidemiol Biomarkers Prev 1999;8:547–51.[Abstract/Free Full Text]
  18. Hildesheim A, Chen CJ, Caporaso NE, et al. Cytochrome P4502E1 genetic polymorphisms and risk of nasopharyngeal carcinoma: results from a case-control study conducted in Taiwan. Cancer Epidemiol Biomarkers Prev 1995;4:607–10.[Abstract]
  19. Hirunsatit R, Kongruttanachok N, Shotelersuk K, et al. Polymeric immunoglobulin receptor polymorphisms and risk of nasopharyngeal cancer. BMC Genet 2003;4:3.[CrossRef][Medline]
  20. Jalbout M, Bouaouina N, Gargouri J, Corbex M, Ben Ahmed S, Chouchane L. Polymorphism of the stress protein HSP70-2 gene is associated with the susceptibility to the nasopharyngeal carcinoma. Cancer Lett 2003;193:75–81.[CrossRef][Medline]
  21. Cho EY, Hildesheim A, Chen CJ, et al. Nasopharyngeal carcinoma and genetic polymorphisms of DNA repair enzymes XRCC1 and hOGG1. Cancer Epidemiol Biomarkers Prev 2003;12:1100–4.[Abstract/Free Full Text]
  22. Xiong W, Zeng ZY, Li XL, et al. Single-nucleotide polymorphisms in NGX6 gene and their correlation with nasopharyngeal carcinoma. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2002;34:512–5.
  23. Huang H, Zhou YF, Jiang PZ, Shen XM, Yao KT. Screening of single nucleotide polymorphisms in nasopharyngeal carcinoma associated genes by denaturing high-performance liquid chromatography. Di Yi Jun Yi Da Xue Xue Bao 2002;22:602–4.[Medline]
  24. Duh FM, Fivash M, Moody M, et al. Characterization of a new SNP c767A/T (Arg222Trp) in the candidate TSG FUS2 on human chromosome 3p21.3: prevalence in Asian populations and analysis of association with nasopharyngeal cancer. Mol Cell Probes 2004;18:39–44.[CrossRef][Medline]
  25. Ren W, Zheng H, Li M, et al. A functional single nucleotide polymorphism site detected in nasopharyngeal carcinoma-associated transforming gene Tx. Cancer Genet Cytogenet 2005;157:49–52.[CrossRef][Medline]
  26. Jia WH, Collins A, Zeng YX. Complex segregation analysis of nasopharyngeal carcinoma in Guangdong, China: evidence for a multifactorial mode of inheritance (complex segregation analysis of NPC in China). Eur J Hum Genet 2005;13:248–52.[CrossRef][Medline]
  27. Coffin CM, Rich SS, Dehner LP. Familial aggregation of nasopharyngeal carcinoma and other malignancies. A clinicopathologic description. Cancer 1991;68:1323–8.[CrossRef][Medline]
  28. Jia WH, Shao JY, Feng BJ, Zeng YX. Genetic component involved in nasopharyngeal carcinoma development. Cancer Reviews:Asia-Pacific 2003;1:31–49.
  29. Jia WH, Feng BJ, Xu ZL, et al. Familial risk and clustering of nasopharyngeal carcinoma in Guangdong, China. Cancer 2004;101:363–9.[CrossRef][Medline]
  30. Feng BJ, Huang W, Shugart YY, et al. Genome-wide scan for familial nasopharyngeal carcinoma reveals evidence of linkage to chromosome 4. Nat Genet 2002;31:395–9.[Medline]
  31. Chen HK, Feng BJ, Liang H, Zhang R, Zeng YX. The susceptibility gene for familial nasopharyngeal carcinoma is mapped on chromosome 4p11-p14 by haplotype analysis. Chinese Science Bulletin 2003;48:2327–30.[CrossRef]
  32. Hillier LW, Graves TA, Fulton RS, et al. Generation and annotation of the DNA sequences of human chromosomes 2 and 4. Nature 2005;434:724–31.[CrossRef][Medline]
  33. Bajic VB, Tan SL, Suzuki Y, Sugano S. Promoter prediction analysis on the whole human genome. Nat Biotechnol 2004;22:1467–73.[CrossRef][Medline]
  34. Christova R, Oelgeschlager T. Association of human TFIID-promoter complexes with silenced mitotic chromatin in vivo. Nat Cell Biol 2002;4:79–82.[CrossRef][Medline]
  35. Humbert O, Achour I, Lautier D, Laurent G, Salles B. hMSH2 expression is driven by AP1-dependent regulation through phorbol-ester exposure. Nucleic Acids Res 2003;31:5627–34.[Abstract/Free Full Text]
  36. Liang H, Chen H, Shen Y, et al. A rare polymorphism of the COX7B2 gene in a Cantonese family with nasopharyngeal carcinoma. Sci China C Life Sci 2004;47:449–53.[CrossRef][Medline]
  37. Ranta S, Zhang Y, Ross B, et al. The neuronal ceroid lipofuscinoses in human EPMR and mnd mutant mice are associated with mutations in CLN8. Nat Genet 1999;23:233–6.[CrossRef][Medline]
  38. Norio R. Finnish disease heritage I: characteristics, causes, background. Hum Genet 2003;112:441–56.[Medline]
  39. Laitinen T, Polvi A, Rydman P, et al. Characterization of a common susceptibility locus for asthma-related traits. Science 2004;304:300–4.[Abstract/Free Full Text]
  40. Xu J, Zheng SL, Komiya A, et al. Germline mutations and sequence variants of the macrophage scavenger receptor 1 gene are associated with prostate cancer risk. Nat Genet 2002;32:321–5.[CrossRef][Medline]
  41. Brennan P, Floettmann JE, Mehl A, Jones M, Rowe M. Mechanism of action of a novel latent membrane protein-1 dominant negative. J Biol Chem 2001;276:1195–203.[Abstract/Free Full Text]
  42. Hatzivassiliou E, Mosialos G. Cellular signaling pathways engaged by the Epstein-Barr virus transforming protein LMP1. Front Biosci 2002;7:d319–29.[Medline]
  43. Dirmeier U, Hoffmann R, Kilger E, et al. Latent membrane protein 1 of Epstein-Barr virus coordinately regulates proliferation with control of apoptosis. Oncogene 2005;24:1711–7.[CrossRef][Medline]
  44. Kondo S, Wakisaka N, Schell MJ, et al. Epstein-Barr virus latent membrane protein 1 induces the matrix metalloproteinase-1 promoter via an Ets binding site formed by a single nucleotide polymorphism: enhanced susceptibility to nasopharyngeal carcinoma. Int J Cancer 2005;115:368–76.[CrossRef][Medline]
  45. Smith PR, Gao Y, Karran L, Jones MD, Snudden D, Griffin BE. Complex nature of the major viral polyadenylated transcripts in Epstein-Barr virus-associated tumors. J Virol 1993;67:3217–25.[Abstract/Free Full Text]
  46. Broustas CG, Larkins LK, Uhler MD, Hajra AK. Molecular cloning and expression of cDNA encoding rat brain cytosolic acyl-coenzyme A thioester hydrolase. J Biol Chem 1996;271:10470–6.[Abstract/Free Full Text]
  47. Yamada J, Kuramochi Y, Takagi M, Watanabe T, Suga T. Human brain acyl-CoA hydrolase isoforms encoded by a single gene. Biochem Biophys Res Commun 2002;299:49–56.[CrossRef][Medline]
  48. Kuhajda FP. Fatty-acid synthase and human cancer: new perspectives on its role in tumor biology. Nutrition 2000;16:202–8.[CrossRef][Medline]
  49. Alo PL, Visca P, Trombetta G, et al. Fatty acid synthase (FAS) predictive strength in poorly differentiated early breast carcinomas. Tumori 1999;85:35–40.[CrossRef][Medline]
  50. Bull JH, Ellison G, Patel A, et al. Identification of potential diagnostic markers of prostate cancer and prostatic intraepithelial neoplasia using cDNA microarray. Br J Cancer 2001;84:1512–9.[CrossRef][Medline]
  51. Furuya Y, Akimoto S, Yasuda K, Ito H. Apoptosis of androgen-independent prostate cell line induced by inhibition of fatty acid synthesis. Anticancer Res 1997;17:4589–93.[Medline]
  52. Gabrielson EW, Pinn ML, Testa JR, Kuhajda FP. Increased fatty acid synthase is a therapeutic target in mesothelioma. Clin Cancer Res 2001;7:153–7.[Abstract/Free Full Text]
  53. Kridel SJ, Axelrod F, Rozenkrantz N, Smith JW. Orlistat is a novel inhibitor of fatty acid synthase with antitumor activity. Cancer Res 2004;64:2070–5.[Abstract/Free Full Text]
  54. Sista ND, Barry C, Sampson K, Pagano J. Physical and functional interaction of the Epstein-Barr virus BZLF1 transactivator with the retinoic acid receptors RAR {alpha} and RXR {alpha}. Nucleic Acids Res 1995;23:1729–36.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Phil Trans R Soc BHome page
Y. Shen, Q. Xu, Z. Han, H. Liu, and G.-B. Zhou
Analysis of phenotype-genotype connection: the story of dissecting disease pathogenesis in genomic era in China, and beyond
Phil Trans R Soc B, June 29, 2007; 362(1482): 1043 - 1061.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jiang, R.-C.
Right arrow Articles by Zeng, Y.-X.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jiang, R.-C.
Right arrow Articles by Zeng, Y.-X.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online