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Open AcceResearch articleThe claudin gene family: expression in normal and neoplastic tissuesKyle J Hewitt1, Rachana Agarwal1 and Patrice J Morin*1,2

Address: 1Laboratory of Cellular and Molecular Biology, National Institute on Aging, Baltimore MD 21224, USA and 2Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD 21287, USA

Email: Kyle J Hewitt - Hewittkyl@grc.nia.nih.gov; Rachana Agarwal - agarwalra@grc.nia.nih.gov; Patrice J Morin* - morinp@grc.nia.nih.gov

* Corresponding author

AbstractBackground: The claudin (CLDN) genes encode a family of proteins important in tight junctionformation and function. Recently, it has become apparent that CLDN gene expression is frequentlyaltered in several human cancers. However, the exact patterns of CLDN expression in variouscancers is unknown, as only a limited number of CLDN genes have been investigated in a fewtumors.

Methods: We identified all the human CLDN genes from Genbank and we used the large publicSAGE database to ascertain the gene expression of all 21 CLDN in 266 normal and neoplastictissues. Using real-time RT-PCR, we also surveyed a subset of 13 CLDN genes in 24 normal and 24neoplastic tissues.

Results: We show that claudins represent a family of highly related proteins, with claudin-16, and-23 being the most different from the others. From in silico analysis and RT-PCR data, we find thatmost claudin genes appear decreased in cancer, while CLDN3, CLDN4, and CLDN7 are elevated inseveral malignancies such as those originating from the pancreas, bladder, thyroid, fallopian tubes,ovary, stomach, colon, breast, uterus, and the prostate. Interestingly, CLDN5 is highly expressed invascular endothelial cells, providing a possible target for antiangiogenic therapy. CLDN18 mightrepresent a biomarker for gastric cancer.

Conclusion: Our study confirms previously known CLDN gene expression patterns and identifiesnew ones, which may have applications in the detection, prognosis and therapy of several humancancers. In particular we identify several malignancies that express CLDN3 and CLDN4. Thesecancers may represent ideal candidates for a novel therapy being developed based on CPE, a toxinthat specifically binds claudin-3 and claudin-4.

BackgroundThe claudin family consists of approximately 23 proteinsthat are essential for the formation of tight junctions (TJs)in epithelial and endothelial cells [1]. TJs have crucialroles in the control of paracellular transport and in themaintenance of cell polarity. It is thought that variousclaudin family members can confer different properties to

epithelial cell permeability and account for some of theselective variability of different barriers [1]. Indeed, mosttissues express multiple claudins, which can interact inboth homotypic and heterotypic fashion to form the tightjunction strands. The exact combination of claudin pro-teins within a given tissue is thought to determine theselectivity and strength of the tight junctions. Underscor-

Published: 12 July 2006

BMC Cancer 2006, 6:186 doi:10.1186/1471-2407-6-186

Received: 27 April 2006Accepted: 12 July 2006

This article is available from: http://www.biomedcentral.com/1471-2407/6/186

© 2006 Hewitt et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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BMC Cancer 2006, 6:186 http://www.biomedcentral.com/1471-2407/6/186

ing the critical roles of claudin proteins are recent obser-vations that germline mutation in these genes can lead tovarious familial diseases, such as neonatal sclerosingcholangitis (CLDN1) [2], nonsyndromic recessive deaf-ness (CLDN14) [3], and familial hypomagnesaemia(CLDN16)[4].

Recent gene expression profiling analyses have shown thatclaudin gene expression is frequently altered in variouscancers (reviewed in [5,6]). For example, CLDN3, andCLDN4 have been found frequently up-regulated in ovar-ian, breast, prostate and pancreatic tumors [7-11]. CLDN7has been found downregulated in breast and head andneck cancer, but elevated in stomach cancer [12,13].CLDN1 is typically downregulated in various cancers, buthas also been reported to be elevated. The picture thatemerges suggests that claudin expression is altered in sev-eral human tumors. Specifically, CLDN1,3,4,5,7,10,16have been found altered in various cancers [5]. The over-expression of these proteins in cancer (which typicallylose their TJs) is unexpected but may be related to rolesthat are unrelated to TJ formation [5]. Indeed, recent worksuggests that claudins may be involved in survival andinvasion of cancer cells [12,14,15].

Regardless of their exact functions in cancer cells, claudinprotein expression may have significant clinical relevance[5,6]. For example, claudin-1 expression has been shownto have prognostic value in colon cancer [16], claudin-18in gastric cancer [17], and claudin-10 in hepatocellularcarcinoma [18]. In addition, because claudins are surfaceproteins, they may represent useful target for various ther-apeutic strategies. Of particular interest, in the possibleuse of Clostridium perfringens enterotoxin (CPE) as a novelchemotherapeutic compound. CPE is a natural ligand forclaudin-3 and -4 proteins, and binding of the toxin tothese claudins leads to a rapid cytolysis of the cells [19].Recent preclinical studies have suggested that CPE may beeffective against claudin-3 and -4-expressing malignancies[8,9,11,20].

Unfortunately, the exact patterns of expression of the var-ious claudins in different cancers and normal tissues arenot well known. To date, only a few of the claudin pro-teins have been investigated in a relatively limited numberof cancers. In this report, we use the vast amount of datapresent in the public SAGE database to create a claudingene expression profile of all the known claudin genes, ina large number of tissues. We then survey a subset of theseclaudin genes using real-time RT-PCR in a panel of nor-mal and neoplastic tissues. Our study confirms previousclaudin gene expression patterns and identifies new ones,which may potentially be of clinical use for various can-cers.

MethodsClaudin homology and phylogenetic tree21 human claudin genes and corresponding proteinssequences were identified and downloaded from Gen-Bank. The ClustalW software (with the Blosum62 matrix)was used to produce a multiple sequence alignment of allthese human claudin protein sequences and the Jalviewsoftware was then used to visualize the results [21]. A phy-logenetic tree of the claudin proteins was produced withClustalW. The clustalW phylogenetic calculations arebased on the neighbor-joining method of Saitou and Nei[22].

In silico analysis of claudin gene expressionMining of the SAGE Genie database [23] for libraries thatexpressed GAPDH or ACTB yielded a total of 266 SAGElibraries with at least some level of expression of thesecontrol genes. These libraries were then examined for theexpression of all 21 human CLDN genes that we identi-fied. SAGE data for both normal and cancerous tissueswas exported to an excel spreadsheet, and expression lev-els converted to tags per 200,000 (the complete dataset isavailable as additional file 1). The dChip software http://www.dchip.org program was then used to visualize thisdata, assigning darker shades of red to higher number oftags.

Real-time RT-PCR of claudin family membersA total of 48 cDNA preparations from various normal andneoplastic tissues (24 each) were purchased from Bio-chains (Hayward, CA). The GeneAmp 7300 SequenceDetection System (PE Applied Biosystems) was used fordetecting RT-PCR products in real-time with the SYBRGreen I assay, as previously described [24]. The primersfor the various CLDN genes(CLDN1,2,3,4,5,7,8,9,10,11,12,16,18) and the controlGAPDH were designed to cross intron-exon boundaries todistinguish PCR products generated from genomic versuscDNA template. For CLDN genes lacking introns, real-time RT-PCR was performed by the polyA cDNA-specificRT-PCR method [25]. The primer sequences are availableonline as additional file 2.

Each PCR reaction was optimized to ensure that a singleband of the appropriate size was amplified and that nobands corresponding to genomic DNA amplification orprimer-dimer pairs were present. The PCR cycling condi-tions were performed for all samples as follows: 50°C, 2minutes for AmpErase UNG incubation, 95°C, 10 min-utes for AmpliTaq Gold activation, and 40 cycles for themelting (95°C, 15 seconds) and annealing/extension(60°C for 1 minute) steps. PCR reactions for each tem-plate were done in duplicate in 96-well plates.

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The comparative CT method (PE Applied Biosystems, Fos-ter City, CA) was used to determine relative quantitationof gene expression for each CLDN gene compared to theGAPDH control. First, the CT values from GAPDH reac-tions were averaged for each duplicate. Next, the relativedifference between GAPDH and each duplicate was calcu-lated as previously described [24]. The final values werethen averaged for each duplicate set, and used in thedChip analysis. Clustering of the CLDN genes was per-formed with distances based on 1-rank correlation andthe centroid linkage method.

ResultsThe claudin family of proteinsAs a starting point for our analyses, we identified 21 dif-ferent human claudin proteins in the GenBank database.Alignment of these 21 sequences using ClustalW showsthat most of the claudin proteins are extremely similar,especially in the membrane-spanning regions (Figure 1A).Notable exceptions are claudin-16, which contains a 66 aaextension at the N-terminus, claudin-18, which has anextension in the second extracellular loop, and claudin-23with a longer C-terminal tail. A phylogenetic tree was alsogenerated to better identify similar members of the family.Overall, the tree demonstrated that the claudins constitutehighly related family of proteins, with claudin-16, and -23being the most different from the others (Figure 1B).Claudin-6 and -9 are the most similar, followed by clau-din-3 and -4, and claudin-1 and -7.

In silico analysis of CLDN expression in 266 tissuesBecause serial analysis of gene expression (SAGE) meas-ures absolute levels of transcripts, cross-comparison ofSAGE data is possible across experiments and laboratories[26]. The SAGE Genie database has been developed toallow in silico analysis of gene expression and comparisonof transcript levels in a large number of normal and dis-eased tissues [23]. Using the SAGE Genie database, weextracted gene expression data for all 21 human claudinsacross 266 tissues (Figure 2 and additional file 1). Someof these genes, such as CLDN1,2,3,4,5,7,11,12, and 15 areexpressed in a large number of different tissues. In con-trast, other claudins, such as CLDN14,16,17,20, and 22have much more restricted expression patterns. CLDN17,for example, was found expressed in only one SAGElibrary (normal kidney), at low levels. CLDN20 was onlyfound in 3 cancer libraries total (a chondrosarcoma, abrain cancer, and a liver tumor). Similarly, CLDN22 wasonly found in 2 breast cancer libraries and one brainastrocytoma library. On the other hand, CLDN3,4, and 7were highly expressed in most normal epithelial cells aswell as their corresponding neoplasias. Further analysissuggested that these claudins are frequently elevated incancer. For example, CLDN3 was elevated in tumors of thelung, prostate, breast, kidney, and ovary compared to

their normal counterparts. Similarly, CLDN4 was elevatedin tumors of the lung, breast, stomach, pancreas, andovary. CLDN7 was elevated in cancers of the thyroid, lung,stomach, pancreas, liver, kidney, and ovary. Interestingly,CLDN6 was frequently expressed in embryonic stem cells(ESCs) but generally not in other tissues. CLDN7 was theonly other CLDN expressed at significant level in ESCs.CLDN18 expression seemed to be mostly restricted to thestomach and the lungs. Vascular endothelial cellsexpressed CLDN5 at high levels, suggesting a new targetfor antiangiogenic therapy. Brain had distinctive claudinexpression profiles, with CLDN3,4 and 7, expressed at lowlevels but CLDN2 and CLDN5 very highly expressed. Thispattern was opposite to what we observed in epithelialcells. CLDN12 was the most widely expressed gene andappeared expressed constitutively in most tissues.

Real-time RT-PCR analysis of CLDN expressionIn order to validate and extend the in silico resultsobtained with the SAGE Genie database, we performedreal-time RT-PCR analysis on a subset of CLDN genes (13genes total) to survey gene expression in several tissues.Gene-specific primers forCLDN1,2,3,4,5,7,8,9,10,11,12,16,18 were designed andoptimized. These CLDN genes were chosen because theywere expressed at detectable levels in several tissues andrepresented a wide variety of different expression patternsas suggested by the SAGE database analysis. A total of 24normal tissues and 24 tumors were surveyed for CLDNexpression (Figure 3). Using this technique, we find thatthe various normal tissues express a wide variety of CLDNgenes (Figure 3A). For example, the kidney expresses highlevels of CLDN10 and CLDN12, but also expresses somelevels of all the other CLDN genes tested (with the excep-tion of CLDN18). Clustering of CDLN genes showed atight association of CLDN3,4,7 in terms of their expres-sion patterns, suggesting a coordinate regulation of thesegenes. The CLDN3,4,7 cluster was found expressed at highlevels in normal pancreas, salivary gland, kidney, adrenalgland, small intestine, colon, and thyroid. Examination ofthe tumor samples revealed that the diversity of CLDNexpression was decreased in these samples (Figure 3B).Except for the CLDN3,4,7 cluster, which was also presentand often elevated in tumors, the other CLDNs appearedto be expressed at relatively low levels. CLDN3,4,7 wereexpressed in tumors of the pancreas, bladder, thyroid, fal-lopian tubes, ovary, stomach, colon, breast, uterus, andprostate. Because of the low number of samples exam-ined, this survey doe not represent an exhaustive analysisof CLDN gene expression in various tissues but rather aninitial study of tissue specificity. However, the similarityin the gene expression patterns identified through this sur-vey and previously known patterns (for CLDN3 and 4, forexample) is striking. Additional studies with several

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Multiple alignment and family tree of claudin proteinsFigure 1Multiple alignment and family tree of claudin proteins. A. ClustalW was used to generate alignment of all the human claudin protein sequences and the residues were then colored using Jalview [21] according to amino acid conservation among family members. Red bars underneath alignment indicate predicted transmembrane domains. The level of conservation and the con-sensus are also shown below the alignment. B. Phylogenetic tree of claudin family members. The tree was generated using ClustalW and visualize in Jalview. The numbers on the branches are Blosum62 scores, indicating distances among family mem-bers.

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In silico analysis of claudin gene expression in various normal and neoplastic tissuesFigure 2In silico analysis of claudin gene expression in various normal and neoplastic tissues. 266 SAGE libraries were examined for the expression of all 21 CLDN genes using SAGE Genie [23]. SAGE data was compiled from both normal and cancerous tissue, and analyzed using dChip software http://www.dchip.org program, which assigned darker shades of red to higher number of tags. GAPDH and ACTN levels were also analyzed as controls. While some CLDN genes are ubiquitously expressed, others exhibit highly tissue specific patterns. A detailed spreadsheet of the data is available as supplementary information.

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Brain_fetal_normal_B_S1Brain_normal_cerebellum_B_1Brain_normal_cortex_B_BB542Brain_normal_cortex_B_pool6Brain_astrocyte_normal_CL_NHA5Brain_normal_cerebellum_B_BB542Brain_normal_leptomeninges_B_AL2Brain_normal_peds_cortex_B_H1571Brain_normal_thalamus_B_1Brain_normal_substantia_nigra_B_1Brain_astrocytoma_grade_II_B_H359Brain_astrocytoma_grade_II_B_H501Brain_astrocytoma_grade_III_B_407Brain_astrocytoma_grade_III_B_H970Brain_ependymoma_B_1394Brain_astrocytoma_grade_III_B_439Brain_glioblastoma_B_R70Brain_glioblastoma_CL_H392Brain_astrocytoma_grade_II_B_H388Brain_astrocytoma_grade_III_B_343Brain_astrocytoma_grade_III_B_828Brain_astrocytoma_grade_III_B_H1055Brain_astrocytoma_grade_III_B_R140Brain_ependymoblastoma_B_819Brain_ependymoma_B_239Brain_glioblastoma_B_H1110Brain_glioblastoma_B_H1353Brain_glioblastoma_B_H1425CBrain_glioblastoma_B_pooledBrain_glioblastoma_CL_H54+EGFRvIIIBrain_glioblastoma_CL_H54+LacZBrain_glioblastoma_CS_TA2Brain_medulloblastoma_B_98-09-P558Brain_medulloblastoma_B_98-13-P301Brain_medulloblastoma_B_DL5Brain_medulloblastoma_B_H484Brain_medulloblastoma_CL_H341Brain_medulloblastoma_B_C609Brain_medulloblastoma_B_98-05-P040Brain_ependymoma_B_R455Brain_astrocytoma_grade_II_B_H530Brain_oligodendroglioma_B_H988Brain_astrocytoma_grade_III_B_H1020Brain_medulloblastoma_B_97-05-P015Brain_medulloblastoma_B_98-05-P608Brain_medulloblastoma_B_H1322Brain_astrocytoma_grade_I_B_H1043Brain_astrocytoma_grade_II_B_H127Brain_astrocytoma_grade_II_B_H516Brain_astrocytoma_grade_II_B_H518Brain_astrocytoma_grade_II_B_H563Brain_astrocytoma_grade_III_B_584Brain_astrocytoma_grade_III_B_H272Brain_astrocytoma_grade_III_B_R927Brain_ependymoma_B_1150Brain_ependymoma_B_H580Brain_ependymoma_B_R1023Brain_ependymoma_B_R353Brain_ependymoma_B_R510Brain_ependymoma_B_R512Brain_ependymoma_B_R582Brain_ependymoma_B_R628Brain_glioblastoma_B_GBM1062Brain_glioblastoma_B_H1371Brain_glioblastoma_B_H833Brain_glioblastoma_B_R20Brain_glioblastoma_B_R336Brain_glioblastoma_control_CL_H247Brain_glioblastoma_hypoxia_CL_H247Brain_medulloblastoma_B_1273Brain_medulloblastoma_B_96-04-P019Brain_medulloblastoma_B_97-05-P312Brain_medulloblastoma_B_98-04-P117Brain_medulloblastoma_B_DL7Brain_medulloblastoma_B_H1413Brain_medulloblastoma_B_H275Brain_medulloblastoma_B_H306Brain_medulloblastoma_B_H876Brain_medulloblastoma_B_H972Brain_medulloblastoma_CL_D283Brain_medulloblastoma_CL_mhh-1Brain_meningioma_grade_II_B_SF2176Brain_meningioma_grade_II_B_SF3622Brain_meningioma_grade_III_B_SF2366Brain_oligodendroglioma_B_1001Brain_medulloblastoma_CL_4-HCRBrain_meningioma_grade_I_B_SF4271Brain_meningioma_grade_I_B_SF4465Brain_medulloblastoma_B_98-04-P494Lung_normal_B_1Lung_normal_CL_L16Lung_normal_CL_L15Lung_tumor_associated_chronic_inflammation_B_UIHMLung_adenocarcinoma_MD_L10Lung_adenocarcinoma_B_1Lung_adenocarcinoma_MD_L9Breast_normal_FS_NERBreast_normal_B_hyperplasia1Breast_normal_myoepithelium_AP_myoepithelial1Breast_normal_stroma_AP_1Breast_normal_epithelium_AP_1Breast_normal_epithelium_AP_Br_NBreast_normal_organoid_BBreast_carcinoma_associated_stroma_B_IDC7Breast_carcinoma_B_DCIS-5Breast_carcinoma_CL_ZR75_1_tamoxifenBreast_carcinoma_MD_DCISBreast_carcinoma_metastasis_B_2Breast_metastatic_carcinoma_B_95-260Breast_normal_myoepithelium_AP_IDC7Breast_carcinoma_CL_MCF7estradiol_10HBreast_carcinoma_CL_MCF7estradiol_3hBreast_carcinoma_epithelium_AP_T392303Breast_carcinoma 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tumors, including various subtypes, grades, stages, andother clinical parameters will be necessary.

DiscussionAlterations in the expression levels of tight junction pro-teins, especially claudins, continue to be reported in sev-eral cancers. However, an overall view of claudin geneexpression in normal and cancer tissues has been lacking.In this report, we first use the large public SAGE databaseto investigate claudin expression of the 21 human CLDNgenes we have identified in GenBank. We find that, whilesome CLDN genes are ubiquitously expressed, the major-ity of these genes exhibit a very restricted expression pat-tern. CLDN14,16,17,19,20, and 22, for example, arefound in only a few rare libraries. Others such asCLDN3,4,5,7,11, and 12 are much more widely expressed.Our analysis allows for the identification of generalexpression patterns, such as the high expression of

CLDN3,4 and 7 in epithelial tissues, and lower expressionin other tissues, such as the brain.

Our data also reveal claudin expression patterns that werenot previously known and that may have clinical implica-tions. According to our data, gastric cells (both normaland neoplastic) express high levels of CLDN18, whileother tissues do not express this gene. Interestingly, arecent study shows that claudin-18 is highly expressed innormal gastric cells and that this high expression isretained in approximately half the gastric tumors [17].Because of its highly restricted pattern, claudin-18 maytherefore represent a useful target for therapy of gastriccancer, especially in those tumors that maintain high lev-els of this gene. Claudin-18 is likely involved in TJ forma-tion in normal gastric cells, while cancer cells, whichtypically do not form TJ's, may have a more available formof claudin-18. Therefore cancer cells may be more sensi-

Real-time RT-PCR survey of CLDN gene expression in normal and neoplastic tissusFigure 3Real-time RT-PCR survey of CLDN gene expression in normal and neoplastic tissus. Expression data of 13 selected CLDN family members from 24 normal and 24 tumor tissue cDNAs. The dChip software was used to analyze and cluster the data. Cluster-ing of the CLDN genes and the tissues were performed with distances based on 1-rank correlation and the centroid linkage method. Darker shades of red indicate higher CLDN expression.

A B

NORMAL TUMORS

CLD

N5

CLD

N1

CLD

N16

CLD

N18

CLD

N11

CLD

N12

CLD

N8

CLD

N9

CLD

N10

CLD

N2

CLD

N3

CLD

N4

CLD

N7

t-Braint-Lymph. Hodgkinst-Pancreast-Bladdert-Skint-Thyroidt-Fallopian Tubet-Ovaryt-Stomacht-Colont-Kidneyt-Breastt-Lungt-Uterust-Lymph. Non-Hodgkinst-Prostatet-Esophagust-Skeletal Musclet-Bonet-Testist-Livert-Neurilemmomat-Lymphomat-Skin Melanoma

CLD

N8

CLD

N3

CLD

N4

CLD

N7

CLD

N11

CLD

N10

CLD

N12

CLD

N18

CLD

N1

CLD

N2

CLD

N16

CLD

N5

CLD

N9

n-Musclen-Brainn-Heartn-Stomachn-Testisn-Salivaryn-Pancreasn-Bone Marrown-Kidneyn-Adrenal Glandn-Livern-Fetal Livern-Ovaryn-Uterusn-Fetal Brainn-Spleenn-Small Intestinen-Colonn-Thyroidn-PBLn-Placentan-Prostaten-Lungn-Skin

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tive to therapy involving the targeting of this molecule.We also find that CLDN5 is not generally expressed in epi-thelial tissues but is expressed at high levels in all vascularendothelial cell libraries analyzed. Although alsoexpressed in the brain, CLDN5 may represent a target forantiangiogenic therapy, especially if using compoundsthat cannot cross the blood-brain barrier.

Our RT-PCR experiments provide a more quantitativelook at claudin gene expression in several normal andneoplastic tissues. It is important to note that these RT-PCR investigations do not represent an exhaustive studyof CLDN gene expression, but rather a survey of expres-sion in a large number of different human tissues. Follow-up studies on multiple samples for these different malig-nancies will be necessary to clearly establish the extentand levels of expression of claudins in these tissues. How-ever, it is important to note that the patterns of geneexpression obtained by real-time RT-PCR (Figure 3)closely mirrors the in silico findings using the SAGE Geniedatabase (Figure 2). For example, the CLDN3 and CLDN4expression patterns are consistent between the two analy-ses (as described above). In addition, we also observe highcorrespondence in the two approaches when examiningCLDN5 expression, which appears to be especially high innormal brain and brain cancer. Interestingly, when clus-tering our RT-PCR data for gene expression patterns, wefind CLDN3,4,7 are very similar in their expression, sug-gesting coordinate regulation. The fact that theCLDN3,4,7 cluster is present in both normal and tumorssuggests that the mechanisms that lead to the coordinatedexpression of claudins in normal cells is conserved intumor cells, although it may be inappropriately activatedin cancer. It will certainly be interesting to elucidate themechanisms that lead to the inappropriate activation ofthese genes.

Our in silico and RT-PCR results are consistent withnumerous previous reports showing that CLDN3 andCLDN4 are overexpressed in breast [11], ovarian [7], andprostate tumors [9]. In addition, our data showing overex-pression of CLDN4 in pancreatic cancer is also in agree-ment with previous reports [8,27]. The finding ofexpression of these claudins in other tumors, such as blad-der, thyroid, fallopian tubes, stomach, colon, and uterus,is novel and warrants further investigation. CPE-basedtherapy, which specifically targets cells expressing clau-din-3 or claudin-4 [8,9,11,20], may be worth exploring inthese malignancies as well. The fact that CLDN3, andCLDN4 are expressed in several normal tissues (Figure 3A)certainly suggests that systemic administration of CPEmay have significant toxic effects. However, the therapeu-tic index of this compound will depend on the level of up-regulation in the various tumors under study and themode of administration. In ovarian cancer, for example,

where both CLDN3 and CLDN4 are highly up regulatedand where intraperitoneal therapy is possible, CPE treat-ment is certainly an interesting possibility.

In this report we study the expression of the CLDN genesat the mRNA level, but it will obviously be essential to val-idate these findings at the protein level when all the anti-bodies are available, as posttranslational mechanismshave been shown to regulate claudin protein levels andlocalization [5]. In addition, it will be important to inves-tigate the various claudins studied here for their potentialclinical use in cancer therapy and diagnosis. With over 20known members, many of which, as we show in thisreport, exhibit high tissue-specific expression and deregu-lation in various cancers, the claudin family of membraneproteins may represent ideal targets for cancer diagnosisand therapy.

ConclusionSystematic analysis of CLDN gene expression using in sil-ico and RT-PCR approaches demonstrate a wide range ofexpression patterns among the various claudins in humancancer. CLDN3, CLDN4, and CLDN7 are elevated in sev-eral malignancies such as those originating from the pan-creas, bladder, thyroid, fallopian tubes, ovary, stomach,colon, breast, uterus, and the prostate. These cancers arethus ideal candidates for a for a novel therapy being devel-oped based on CPE, a toxin that specifically binds clau-din-3 and claudin-4. CLDN18 is specifically expressed ingastric cells and may represent a marker for gastric tumors.CLDN5 is highly expressed in vascular endothelial cells,providing a possible target for antiangiogenic therapy.Overall, a better knowledge of claudin expression in nor-mal and neoplastic tissues may provide new opportunitiesfor the detection, prognosis and therapy of several humancancers.

AbbreviationsTJ: tight junction; CPE, Clostridium perfringens enterotoxin;RT-PCR, reverse-transcriptase PCR; SAGE: serial analysisof gene expression.

Competing interestsThe author(s) declare that they have no competing inter-ests.

Authors' contributionsKJH: Performed RT-PCR experiments, multiple align-ments, and drafted a first draft of the manuscript; RA:Designed and tested some of the primers for the RT-PCRexperiments; PJM: Designed and coordinated the study,interpreted the data, and drafted the manuscript.

AcknowledgementsWe thank Drs Hiroshi Honda, Ashani Weeraratna, Theresa D'Souza, Jiang-hong Li, and Cheryl Sherman-Baust for useful comments on the manuscript.

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