Littérature scientifique sur le sujet « CLDN19 »

Despite recent improvements in diagnostic ability and treatment strategies, advanced gastric cancer (GC) has a high frequency of recurrence and metastasis, with poor prognosis. To improve the treatment results of GC, the search for new treatment targets from proteins related to epithelial–mesenchymal transition (EMT) and cell–cell adhesion is currently being conducted. EMT plays an important role in cancer metastasis and is initiated by the loss of cell–cell adhesion, such as tight junctions (TJs), adherens junctions, desmosomes, and gap junctions. Among these, claudins (CLDNs) are highly expressed in some cancers, including GC. Abnormal expression of CLDN1, CLDN2, CLDN3, CLDN4, CLDN6, CLDN7, CLDN10, CLDN11, CLDN14, CLDN17, CLDN18, and CLDN23 have been reported. Among these, CLDN18 is of particular interest. In The Cancer Genome Atlas, GC was classified into four new molecular subtypes, and CLDN18–ARHGAP fusion was observed in the genomically stable type. An anti-CLDN18.2 antibody drug was recently developed as a therapeutic drug for GC, and the results of clinical trials are highly predictable. Thus, CLDNs are highly expressed in GC as TJs and are expected targets for new antibody drugs. Herein, we review the literature on CLDNs, focusing on CLDN18 in GC.

ABSTRACT Hepatitis C virus (HCV) is a global challenge to public health. Several factors have been proven to be critical for HCV entry, including the newly identified claudin-1 (CLDN1). However, the mechanism of HCV entry is still obscure. Presently, among the 20 members of the claudin family identified in humans so far, CLDN1 has been the only member shown to be necessary for HCV entry. Recently, we discovered that Bel7402, an HCV-permissive cell line, does not express CLDN1 but expresses other members of claudin family. Among these claudins, CLDN9 was able to mediate HCV entry just as efficiently as CLDN1. We then examined if other members of the claudin family could mediate entry. We show that CLDN6 and CLDN9, but not CLDN2, CLDN3, CLDN4, CLDN7, CLDN11, CLDN12, CLDN15, CLDN17, and CLDN23, were able to mediate the entry of HCV into target cells. We found that CLDN6 and CLDN9 are expressed in the liver, the primary site of HCV replication. We also showed that CLDN6 and CLDN9, but not CLDN1, are expressed in peripheral blood mononuclear cells, an additional site of HCV replication. Through sequence comparison and mutagenesis studies, we show that residues N38 and V45 in the first extracellular loop (EL1) of CLDN9 are necessary for HCV entry.

The thick ascending limb (TAL) of Henle’s loop drives paracellular Na+, Ca2+, and Mg2+ reabsorption via the tight junction (TJ). The TJ is composed of claudins that consist of four transmembrane segments, two extracellular segments (ECS1 and -2), and one intracellular loop. Claudins interact within the same (cis) and opposing (trans) plasma membranes. The claudins Cldn10b, -16, and -19 facilitate cation reabsorption in the TAL, and their absence leads to a severe disturbance of renal ion homeostasis. We combined electrophysiological measurements on microperfused mouse TAL segments with subsequent analysis of claudin expression by immunostaining and confocal microscopy. Claudin interaction properties were examined using heterologous expression in the TJ-free cell line HEK 293, live-cell imaging, and Förster/FRET. To reveal determinants of interaction properties, a set of TAL claudin protein chimeras was created and analyzed. Our main findings are that (i) TAL TJs show a mosaic expression pattern of either cldn10b or cldn3/cldn16/cldn19 in a complex; (ii) TJs dominated by cldn10b prefer Na+ over Mg2+, whereas TJs dominated by cldn16 favor Mg2+ over Na+; (iii) cldn10b does not interact with other TAL claudins, whereas cldn3 and cldn16 can interact with cldn19 to form joint strands; and (iv) further claudin segments in addition to ECS2 are crucial for trans interaction. We suggest the existence of at least two spatially distinct types of paracellular channels in TAL: a cldn10b-based channel for monovalent cations such as Na+ and a spatially distinct site for reabsorption of divalent cations such as Ca2+ and Mg2+.

Euryhaline teleost kidneys undergo a major functional switch from being filtratory in freshwater (FW) to being predominantly secretory in seawater (SW) conditions. The transition involves both vascular and tubular effects. There is consensus that the glomerular filtration rate is greatly reduced upon exposure to hyperosmotic conditions. Yet, regulation at the tubular level has only been examined sporadically in a few different species. This study aimed to obtain a broader understanding of transcriptional regulation in proximal versus distal tubular segments during osmotic transitions. Proximal and distal tubule cells were dissected separately by laser capture microdissection, RNA was extracted, and relative mRNA expression levels of >30 targets involved in solute and water transport were quantified by quantitative PCR in relation to segment type in fish acclimated to FW or SW. The gene categories were aquaporins, solute transporters, fxyd proteins, and tight junction proteins. aqp8bb1, aqp10b1, nhe3, sglt1, slc41a1, cnnm3, fxyd12a, cldn3b, cldn10b, cldn15a, and cldn12 were expressed at a higher level in proximal compared with distal tubules. aqp1aa, aqp1ab, nka-a1a, nka-a1b, nkcc1a, nkcc2, ncc, clc-k, slc26a6C, sglt2, fxyd2, cldn3a, and occln were expressed at a higher level in distal compared with proximal tubules. Expression of aqp1aa, aqp3a1, aqp10b1, ncc, nhe3, cftr, sglt1, slc41a1, fxyd12a, cldn3a, cldn3b, cldn3c, cldn10b, cldn10e, cldn28a, and cldn30c was higher in SW- than in FW-acclimated salmon, whereas the opposite was the case for aqp1ab, slc26a6C, and fxyd2. The data show distinct segmental distribution of transport genes and a significant regulation of tubular transcripts when kidney function is modulated during salinity transitions.

Claudins are integral proteins expressed at the tight junctions of epithelial and endothelial cells. In the mammalian kidney, every tubular segment express a specific set of claudins that give to that segment unique properties regarding permeability and selectivity of the paracellular pathway. So far, 3 claudins (10b, 16 and 19) have been causally traced to rare human syndromes: variants of CLDN10b cause HELIX syndrome and variants of CLDN16 or CLDN19 cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis. The review summarizes our current knowledge on the physiology of mammalian tight junctions and paracellular ion transport, as well as on the role of the 3 above-mentioned claudins in health and disease. Claudin 14, although not having been causally linked to any rare renal disease, is also considered, because available evidence suggests that it may interact with claudin 16. Some single-nucleotide polymorphisms of CLDN14 are associated with urinary calcium excretion and/or kidney stones. For each claudin considered, the pattern of expression, the function and the human syndrome caused by pathogenic variants are described.

Abstract Introduction Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare disorder caused by perturbation in renal reabsorption of magnesium and calcium. Biallelic pathogenic variants either in gene CLDN16 or CLDN19 are responsible for molecular defects. Most patients with CLDN19 variants have been associated with ocular involvements (FHHNCOI). Patient and Methods We had a pediatric patient with hypercalciuric hypomagnesemia and bilateral chorioretinal atrophy. Metabolic profiling and radiology examinations were performed, in addition to whole exome sequencing (WES) used for detection of the causative variant. Results Analysis of WES revealed a homozygous c.223G > A (p.G75S) variant in CLDN19. MutationTaster and Combined Annotation-Dependent Depletion support its deleterious effect and SHERLOC's criteria put it in pathogenic category. This variant is previously reported in compound heterozygous state with other known pathogenic variant. As far as we know, it is the first report of this variant in homozygous state. Conclusion The variant found in our patient is pathogenic and compatible with FHHNCOI characteristics. WES is an advantageous tool in molecular diagnosis and finding genetic pathology of this disease. In line with other reports, ocular abnormalities are variable in patients with CLDN19 mutations, and chronic kidney disease and retinal damages must be considered in this group.

1070 Background: Triple negative breast cancer (TNBC) often grows rapidly and has poor prognosis, with a high recurrence rate. Because conventional endocrine treatment and HER2 targeted therapy for TNBC is invalid, chemotherapy is the only systemic treatment for TNBC. It is known that several subtypes within the TNBC show different responses to chemotherapy, depending on the subtypes. Recently, a claudin (CLDN) low breast cancer has been identified, exhibiting low expressions of CLDNs 1, 3, 4 and 7. CLDNs are transmembrane proteins that seal tight junctions and are critical for maintaining cell-to-cell adhesion in epithelial cell sheets. However, their role in cancer progression remains largely unexplored. Methods: Surgically removed, formalin-fixed, paraffin-embedded breast cancers from 341 TNBC patients were analyzed to identify CLDN expression.They underwent wide local excision or mastectomy between March, 2004 and December, 2007 at the breast surgery unit of Asan Medical Central Hospital. Results: In our tumor series, we found 45.0% (153/339) of high expressing cases for CLDN1, 57.0% (192/337) for CLDN3, 57.6% (194/337) for CLDN4 and 44.0% (149/339) for CLDN7. Overall, we found 20.5% (70/341) of cases were within the low CLDN expression group and 79.5% (271/341) of tumors were within the high expression group of CLDN1, 3, 4 ,7. Although the high CLDN expression group was significantly associated with positive lymph node status and higher stage, there were no significant differences between CLDN low and high groups in disease free survival (p=0.477) or overall survival (p=0.253). Conclusions: CLDN high tumors are associated with poor prognosis features, but they are not an independent prognostic factor in TNBC patients. However, the mechanisms underlying the different roles of CLDNs in tumorigenesis are largely unclear. Studying the associations of these CLDNs with the TNBC subgroup of breast cancers might provide us with potential diagnostic biomarkers or therapeutic targets for cancer cells.

Mutations in hepatocyte nuclear factor 1β (HNF1β) cause autosomal dominant tubulointerstitial kidney disease (ADTKD-HNF1β), and patients tend to develop renal cysts, maturity-onset diabetes of the young (MODY), and suffer from electrolyte disturbances, including hypomagnesemia, hypokalemia, and hypocalciuria. Previous HNF1β research focused on the renal distal convoluted tubule (DCT) to elucidate the ADTKD-HNF1β electrolyte phenotype, although 70% of Mg2+ is reabsorbed in the thick ascending limb of Henle’s loop (TAL). An important regulator of Mg2+ reabsorption in the TAL is the calcium-sensing receptor (CaSR). This study used several methods to elucidate the role of HNF1β in electrolyte reabsorption in the TAL. HNF1β ChIP-seq data revealed a conserved HNF1β binding site in the second intron of the CaSR gene. Luciferase-promoter assays displayed a 5.8-fold increase in CaSR expression when HNF1β was present. Expression of the HNF1β p.Lys156Glu mutant, which prevents DNA binding, abolished CaSR expression. Hnf1β knockdown in an immortalized mouse kidney TAL cell line (MKTAL) reduced expression of the CaSR and Cldn14 (claudin 14) by 56% and 48%, respectively, while Cldn10b expression was upregulated 5.0-fold. These results were confirmed in a kidney-specific HNF1β knockout mouse, which exhibited downregulation of the Casr by 81%. Cldn19 and Cldn10b expression levels were also decreased by 37% and 83%, respectively, whereas Cldn3 was upregulated by 4.6-fold. In conclusion, HNF1β is a transcriptional activator of the CaSR. Consequently, patients with HNF1β mutations may have reduced CaSR activity in the kidney, which could explain cyst progression and hyperabsorption of Ca2+ and Mg2+ in the TAL resulting in hypocalciuria.

ABSTRACT It is widely recognized that the claudin (Cldn) family of four tetraspan transmembrane proteins is crucial for tight junction assembly and permeability barrier function; however, the precise role of the tail and loop domains in Cldn function is not understood. We hypothesized that the cytoplasmic tail domain of Cldn6 is crucial for membrane targeting and hence epidermal permeability barrier (EPB) formation. To test this hypothesis via a structure-function approach, we generated a tail deletion of Cldn6 (CΔ187) and evaluated its role in epidermal differentiation and EPB formation through its forced expression via the involucrin (Inv) promoter in the suprabasal compartment of the transgenic mouse epidermis. Even though a functional barrier formed, Inv-CΔ187 mice displayed histological and biochemical abnormalities in the epidermal differentiation program and stimulation of epidermal cell proliferation in both the basal and suprabasal compartments of the interfolliclar epidermis, leading to a thickening of the epidermis after 1 week of age that persisted throughout life. Although some membrane localization was evident, our studies also revealed a significant amount of not only Cldn6 but also Cldn10, Cldn11, and Cldn18 in the cytoplasm of transgenic epidermal cells as well as the activation of a protein-unfolding pathway. These findings demonstrate that the overexpression of a tail truncation mutant of Cldn6 mislocalizes Cldn6 and other Cldn proteins to the cytoplasm and triggers a postnatal increase in proliferation and aberrant differentiation of the epidermis, emphasizing the importance of the Cldn tail domain in membrane targeting and function in vivo.

Abstract The tight junction protein Claudin 6 (CLDN6) is differentially expressed on cancer cells with almost no expression in healthy tissue, making it a valuable therapeutic target for many solid tumor cancers. Despite their potential as cancer therapeutics, very few CLDN6 monoclonal antibodies (MAbs) are in development, because MAbs with high affinity and specificity for CLDN6 are difficult to isolate. CLDN6 is structurally complex, with 4 transmembrane domains and 95% extracellular sequence conservation between human and mouse. Achieving MAb specificity for CLDN6 is especially challenging because its extracellular region strongly resembles those of 23 other human CLDN family members. In particular, the widely expressed CLDN9 differs from CLDN6 by only 3 extracellular residues. Using MAb discovery strategies specifically tailored to complex membrane proteins, including the use of virus-like particles (Lipoparticles), divergent species (chicken) immunization, and optimized phage display panning, we isolated 6 rare MAbs that recognize the native structure of CLDN6 with as low as picomolar affinity. The MAbs were screened against a Membrane Proteome Array containing ~6,000 membrane proteins and demonstrated specificity for CLDN6 with minimal cross-reactivity for CLDN9 or other CLDN family members. Epitope mapping using Shotgun Mutagenesis alanine scanning across the 220 residue CLDN6 sequence distinguished the binding sites of the MAbs from clinical-stage benchmarks. Atomic-level epitope mapping using comprehensive site-specific mutagenesis identified the γ carbon on CLDN6 residue Q156 as the critical structural mechanism enabling these MAbs to differentiate between CLDN6 and CLDN9 with high specificity. The CLDN6 MAbs identified here can be used to study CLDN6-positive cancers, including ovarian, endometrial, lung, and testicular cancer, and have the potential to be developed into highly selective therapeutics. Characterization of highly specific CLDN6 MAbs isolated for treatment of solid tumors MAb IM301 IM302 Benchmark (IMAB027, Astellas) VH CDR3 length (Kabat) 18 18 8 CLDN protein binding: Biosensor KD ± error, nM CLDN6 (target) 0.6 ± 0.03 < 0.001 0.5 ± 0.01 CLDN9 No binding No binding 3.6 ± .09 CLDN3 No binding No binding No binding CLDN4 No binding 146 ± 20 153 ± 6 Mouse CLDN6 binding Yes Yes Yes Cyno CLDN6 binding Yes Yes Yes Conformational epitope Yes Yes Yes Epitope topology Yes Yes Yes Critical CLDN6 epitope residues E48, E154, R158 E154, R158 F35, G37, S39 Citation Format: Brad Screnci, Lewis J. Stafford, Trevor Barnes, Kristen Shema, Samantha Gilman, Rebecca Rimkunas, Suzie Al Absi, Tim Phillips, Charles Azuelos, Katherine Slovik, Paige Muprhy, Daniel B. Harmon, Tom Charpentier, Benjamin J. Doranz, Joseph B. Rucker, Ross Chambers. Atomic-level specificity of Claudin 6 monoclonal antibodies isolated for treating solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 318.

L’ipomagnesemia, ipercalciuria e nefrocalcinosi familiare (FHHNC; OMIM 248250) è una tubulopatia renale rara a trasmissione autosomica recessiva, caratterizzata da perdita della funzione di riassorbimento del magnesio, ipercalciuria, nefrocalcinosi, formazione di calcoli renali, ricorrenti infezioni renali e progressiva insufficienza renale (i pazienti sono candidati a trapianto renale). I pazienti sono anche soggetti a spasmi muscolari, convulsioni e anomalie oculari. Sono inoltre stati osservati alti livelli dell’ormone paratiroideo (PTH) nel corso della malattia. Fino ad oggi la FHHNC è stata diagnosticata in circa 200 individui sparsi in tutto il mondo. Nelle famiglie FHHNC finora indagate la sindrome cosegrega con mutazioni nei geni CLDN16 e CLDN19. Questi due geni codificano per due proteine appartenenti alla famiglia delle claudine, le claudine 16 e 19, espresse nelle tight junctions nel tratto ascendente spesso dell’ansa di Henle. Attualmente si hanno poche informazioni sulla struttura e funzione di queste due proteine. È stato però ipotizzato che, interagendo tra loro, formerebbero dei pori attraverso cui passerebbe il magnesio secondo gradiente elettrochiminco. Lo scopo principale di questo studio è stato quello di (1) espandere lo spettro delle mutazioni nei geni CLDN16 e CLDN19 nelle famiglie FHHNC e indagare la loro distribuzione all’interno di popolazioni differenti e diverse aree geografiche, (2) cercare di stabilire una correlazione genotipo-fenotipo nei pazienti affetti dalla patologia e contribuire all’analisi della relazione struttura-funzione delle claudine 16 e 19, (3) identificare altri eventuali geni coinvolti nell’insorgenza della FHHNC, (4) e, poiché recenti studi hanno associato la sovraespressione di CLDN16 con una diminuzione dell’aggressività del carcinoma al seno, indagare se le mutazioni in CLDN16 identificate come causa dell’insorgenza della FHHNC possano influire sulle eventuali capacità antitumorali della proteina. Lo studio presentato in questa tesi di dottorato è stato condotto su 27 famiglie FHHNC non consanguinee, per un totale di 33 pazienti sottoposti ad analisi genetica. Per ciascuno di questi pazienti e per alcuni parenti sani abbiamo raccolto campioni di sangue dai quali sono stati estratti DNA, RNA totale e proteine. Per ciascuno dei 33 pazienti FHHNC l’analisi genetica è iniziata con il sequenziamento diretto dell’intera regione codificante e dei siti di regolazione dello splicing del gene CLDN16. In 12 pazienti abbiamo identificato complessivamente tre nuove mutazioni non senso (R214X, W217X, Y213X), quattro nuove variazioni presumibilmente associabili all’insorgenza della malattia (L116F, C120Y, G239V, E294K) e cinque mutazioni già precedentemente descritte (S110R, N123fs, L145P, R149X, L151F). Per i 21 pazienti FHHNC, risultati negativi per mutazioni nel gene CLDN16, la caratterizzazione molecolare è proseguita con l’analisi della sequenza codificante e dei siti di regolazione dello splicing del gene CLDN19. In 12 pazienti abbiamo identificato complessivamente una mutazione già descritta in precedenza (G20D) e una delezione che interessa gli esoni 1-4 del gene (è il primo caso noto di delezione quasi totale del gene CLDN19). Risultano tuttora in corso la caratterizzazione molecolare di 9 pazienti per i quali non sono state identificate mutazioni associabili all’insorgenza della malattia nei geni CLDN16 e CLDN19 e le analisi per valutare se mutazioni che comportano una completa o parziale scomparsa della funzione di claudina-16 nel rene possano causare anche diminuzione o scomparsa della sua ipotetica capacità antitumorale nelle cellule di tumori del seno. I risultati di questo lavoro espandono lo spettro delle mutazioni patogenetiche di CLDN16 e CLDN19 nei pazienti FHHNC, contribuendo a fornire informazioni relative alla patogenesi della sindrome e allo studio della relazione struttura-funzione nelle claudine 16 e 19. Saranno tuttavia necessari ulteriori approfondimenti sperimentali per comprendere meglio l’effetto di ciascuna mutazione sulla funzionalità delle due proteine.

The tight junctions (TJ), which are located in the apical region between epithelial and endothelial cells, regulate the paracellular diffusion of ions and small molecules and play an important role in maintaining cell polarity, cell-cell integrity, and permeability. In the lung, epithelial cells are attached by TJ structures. They provide a permeable barrier and cell communication. The loss of barrier integrity, which is maintained by the expression of claudins (Cldn), results in cellular permibilization and leads to paracellular diffusion of solutes and harmful molecules. There are 27 known Cldn homologous members in mice and human. Cldn6 is mostly expressed in embryonic stem cells and associated with the programing of epithelial cells during embryo development and lung morphogenesis. In order to test the hypothesis that Cldn6 expression affects lung morphogenesis, we analyzed the expression pattern of Cldn6 during lung ontogenesis to examine cell-specific expression pattern of Cldn6 during each embryonic period in the mouse lung. Also, we assessed transcriptional regulators and control mechanisms that precisely influence Cldn6 expression in pulmonary cells. We discovered that Cldn6 is an important tight junctional component expressed by pulmonary epithelium during lung organogenesis. We found that normal down-regulation of Cldn6 as development proceeds influences differentiation associated with the transition between the embryonic to the alveolar stage. Conditional gain-of-function and loss-of-function experiments in animal models prove to be the most beneficial tool in deciphering the impact of Cldn in organ formation and maintenance. We generated a conditional transgenic mouse that provides the opportunity to genetically up-regulate Cldn6 in distal lung. Our transgenic mouse showed a delay in lung development and down-regulation of transcriptional factors. Cldn6 is both temporally and spatially controlled in the developing lung and its regulation is maintained by critical transcriptional control networks managed by TTF-1. In lung diseases, altered Cldn expression leads to diseases such as COPD, asthma, and ARDS. The tight junctional proteins are differentially regulated by tobacco smoke exposure and Cldn6 is potentially involved as neighboring epithelial cells respond to tobacco smoke. We exposed adult mice to controlled doses of second hand smoke during four days and A-549 cells to 10% CSE for 6 hours. We discovered that mice lungs respond by down-regulating Cldn6 basal levels and impair barrier function. These results reveal that midgestational up-regulation of Cldn6 and its marked down-regulation as development proceeds illustrate the notion that Cldn6 function is important during early programming stages of lung morphogenesis.

Introducción: analizamos la relación entre fracturas y densidad mineral ósea (DMO) y los SNP (polimorfismos de un solo nucleótido) rs3102735 (163 A/G), rs3134070 (245 T/G) y rs2073618 (1181 G/C) de OPG, el SNP rs2277438 SNP de RANKL, el SNP rs7121 (393 T/C) de GNAS1 y del SNP rs219780 del gen CLDN14 en pacientes con HPP (hiperparatiroidismo primario) esporádico. Métodos: reclutamos 298 pacientes con HPP y 328 voluntarios sanos en un estudio transversal. Analizamos historia de fracturas o litiasis renal, parámetros bioquímicos, DMO en columna lumbar, cadera total, cuello femoral y radio distal y genotipado de los SNP mencionados. Resultados: no encontramos diferencias entre los genotipos de ninguno de los SNP estudiados en relación con la frecuencia de fracturas en HPP o en sujetos control. La DMO fue menor en el radio en los HPP homocigotos para el alelo menor en comparación con el resto de grupos en los SNP de OPG (163 A/G) y (245 T/G) pero no en sujetos control. En el resto de los SNP estudiados no encontramos diferencias entre genotipos y DMO en los sujetos con HPP o control excepto en el SNP de OPG (1181 G/C) en sujetos control con mayor DMO lumbar en el grupo CC respecto del GG. Conclusiones: los sujetos con HPP y homocigotos para el alelo menor (GG) en los SNP rs3102735 (163 A/G) y rs3134070 (245 T/G) de OPG tienen menor DMO en el radio distal. El resto de SNP estudiados no parecen influir en la diferente expresión de las manifestaciones óseas del HPP.
Background: we analyze the relationship between fractures and BMD (bone mineral density) and the rs3102735 (163 A/G), rs3134070 (245 T/G) and rs2073618 (1181 G/C) SNPs of the OPG, the rs2277438 SNP of the RANKL, the rs7121 SNP (393 T/C) of GNAS1 and the rs219780 of CLDN14 in patients with sporadic PHPT (primary hyperparathyroidism). Methods: We enrolled 298 Caucasian patients with PHPT and 328 healthy volunteers in a cross-sectional study. We analyzed history of fractures or renal lithiasis, biochemical determinants, BMD measurements in the lumbar spine, total hip, femoral neck and distal radius, and genotyping for the SNPs to be studied. Results: Regarding the frequency of fractures we found no differences between genotypes in any of the SNPs studied in the PHPT or in the control subjects groups. Significant lower BMD in the distal radius was found in the minor allele homozygotes (GG) compared to heterozygotes and major allele homozygotes in both OPG rs3102735 (163 A/G) and OPG rs3134070 (245 T/G) SNPs in those with PHPT but not in control subjects. We found no difference between genotypes of the rest of the SNPs studied in PHPT or control subjects with the exception of SNP OPG rs2073618 (1181 G/C) in control CC subjects which showed higher lumbar BMD than GG ones. Conclusions: Subjects with PHPT and minor homocygote genotype (GG) for the OPG rs3102735 (163 A/G) and OPG rs3134070 (245 T/G) SNPs have lower BMD in the distal radius. All the other SNPs studied do not appear to influence the different expression of HPP in bone.

Bakterielle Toxine stellen eine wirkungsvolle und effektive Alternative zur Therapie von Tumorerkrankungen dar. Das vom Clostridium perfringens Typ A produzierte Clostridium perfringens enterotoxin (CPE) gehört zu der Gruppe der porenbildenden Toxine und weist eine rezeptorspezifische zytotoxische Wirkung auf, welche über die Membranrezeptoren Cldn3 und Cldn4 entfaltet wird. Diese liegen vor allem in Epithelialkarzinomen wie dem Brust-, Prostata-, oder Kolon-, sowie dem Pankreaskarzinom (PK) stark hochreguliert vor. Ziel dieser Arbeit war die Anwendung des neuen selektiven und effizienten „Onkoleaking“ Suizid-Gentherapie Konzepts für die Behandlung von Cldn3 / 4 überexprimierender PK unter Verwendung eines nicht-viralen translations-optimierten CPE exprimierenden Vektors (optCPE). Weiterhin sollte in dieser Arbeit der genaue molekulare Mechanismus der CPE-vermittelten Zytotoxizität in vitro und auch in vivo analysiert werden. Für die in vitro Analysen wurden verschiedene humane PK Zelllinien, Patienten abgeleitete Xenotransplantate (PDX) und deren abgeleiteten Zellen bezüglich ihrer Cldn3 / 4 Expression und Sensitivität sowohl gegenüber rekombinantem CPE (rekCPE) als auch nach optCPE Gentransfer untersucht. Es konnte eine positive Korrelation zwischen der Effizienz CPE vermittelter Zytotoxizität und der Höhe der Cldn3 / 4 Überexpression gezeigt werden. Des Weiteren wurde die Verfügbarkeit und Zugänglichkeit der CPE Rezeptoren für die Toxinbindung als kritischer Faktor für die durch Porenbildung induzierte Zytotoxizität beschrieben. Auch eine detaillierte Analyse verschiedener apoptotischer und nekrotischer Signalwege und deren Schlüsselmoleküle waren vom besonderen Interesse. Von noch größerer Wichtigkeit war jedoch die Anwendbarkeit und der Nachweis der antitumoralen Wirksamkeit der optCPE-basierten Suizid-Gentherapie mit Hilfe des intratumoralen Jet-Injektion Gentransfers in verschiedenen Luziferase-exprimierenden CDX und PDX Modellen des PK. Alle in vivo Studien zeigten eine selektive optCPE vermittelte Verminderung der Tumorvitalität in Verbindung mit Nekrose, die in fast allen Fällen mit einer Reduktion des Tumorvolumens einher ging. Die tierexperimentellen Studien belegen damit die Effektivität der CPE-basierten Gentherapie im Pankreaskarzinom. Mit diesen neu gewonnenen Erkenntnissen zum „Onkoleaking“ Konzept der CPE Suizid-Gentherapie und deren Wirkungsmechanismen sind Kombinationen mit konventionellen Therapien möglich.
Bacterial toxins have evolved to an effective therapeutic option for cancer therapy and numerous studies demonstrated their antitumoral potential. The Clostridium perfringens enterotoxin (CPE), produced by the anaerobic Clostridium perfringes bacteria, is a pore-forming (oncoleaking) toxin, which binds to its receptors claudin-3 and -4 (Cldn3 / 4) and exerts a selective, receptor-dependent cytotoxicity. The transmembrane tight junction proteins Cldn3 and Cldn4 are known CPE receptors and are highly upregulated in several human epithelial cancers such as breast, colon, ovarian and pancreatic cancer. This study aimed at the evaluation of the potential of oncoleaking gene therapy using a non-viral translation optimized CPE vector (optCPE) as a new suicide approach for the treatment of Cldn3  /  4 overexpressing pancreatic cancer (PC) in vitro and in vivo. We demonstrated the successful in vitro use of optCPE gene transfer in a panel of human PC cells and more importantly patient derived PC xenograft (PDX) derived cells. We showed significant reduction of cell viability in all Cldn3 / 4 overexpressing PC cells after optCPE transfection. Furthermore a positive correlation between CPE cytotoxicity and level of claudin expression was shown. We revealed accessibility of CPE receptors for toxin binding as determining for optCPE mediated cytotoxicity. Since investigation of optCPE induced cell death mechanism was of particular interest, detailed analyses of apoptotic and necrotic key players were performed. By this, caspase dependent- and independent apoptosis and necrosis activation after gene transfer was demonstrated, which was dependent on amount of expressed optCPE and accessibility of Cldn. More importantly, this study demonstrated the applicability and antitumoral efficacy of optCPE gene therapy by the non-viral intratumoral jet-injection gene transfer in vivo in different luciferase-expressing CDX and PDX pancreatic cancer models. The animal experiments demonstrated the selective CPE mediated tumor growth inhibition, associated with reduced tumor viability and effective induction of tumor necrosis. This further corroborated the advantages of this novel oncoleaking strategy. With this gain of knowledge about our new oncoleaking concept of suicidal gene therapy and its mechanism of action, novel combinations with conventional therapies are possible to further improve therapeutic efficacy and to overcome resistance in pancreas carcinoma.

碩士
中山醫學大學
生化暨生物科技研究所
95
The mutations or deletions of CLAUDIN 14 (CLDN14) gene are responsible for nonsyndromic deafness DFNB29 in human. Furthermore, studies with Cldn14-null mice indicated such deafness is due to the degeneration of hair cells leading to cation overload. CLDN14 is a member of CLDN family involved in tight junction strand formation. Tight junction contains the barrier and fence functions to passage of ions and molecules through the selectively paracellular pathway. Previously, we had identified 3 mutants of CLDN14 in prelingual nonsyndromic sensorineural deafness, including 2 missense mutations (M18V and D142N) and 1 deletion mutant (with GG nucleotides at 167-168 being deleted to cause frameshift). To investigate the impact of mutations of CLDN14, the localizations of mutants and wild-type CLDN14 were studied by transfections performed on MDCK cells. Our results indicate that CLDN14-D142N protein localized at the plasma membrane and in cytoplasmic compartment, similar to that of CLDN14-WT protein, while CLDN14-M18V and GG deletion mutant proteins were remained and accumulated in the cytoplasma without being transferred to the plasma membrane. These results suggest that mutations of CLDN14, M18V and GG deletion might cause deafness by inhibitng the formation and function of tight junction.

碩士
中山醫學大學
生物醫學科學學系碩士班
100
The mutations or deletions of CLAUDIN 14 (CLDN14) gene are responsible for nonsyndromic deafness DFNB29 in human. Previously, we had identified 3 mutants of CLDN14 in prelingual nonsyndromic sensorineural deafness, including 2 missense mutations (M18V and D142N) and 1 deletion mutant (W56S). We had have 4 CLDN14 MDCK stable cell line including CLDN14WT, CLDN14M18V, CLDN14W56S and CLDN14D142N in the previous studies. In this study, our results indicated that CLDN14M18V and CLDN14W56S mutant proteins were accumulated and remained in the cytoplasm without being transferred to the plasma membrane. In addition, we found a dominant negative effect in CLDN14M18V mutant when co-transfection CLDN14WT and CLDN14M18V. Moreover, we found that CLDN14M18V proteins are co-localized with lysosome. Furtherlly, we found that the accumulated CLDN14M18V protein was increased without CLDN14 mRNA increased and the molecular size of accumulated CLDN14M18V protein was decreased after treating with chloroquine. In contrast, CLDN14D142N protein localized at the plasma membrane and in cytoplasmic compartment, similar to that of CLDN14WT protein. However, we found that CLDN14 D142N retained its ability in trafficking, but lost significantly its function as a barrier of tight junction in the transepithelial electrical resistance (TER) assay. In addition,the barrier function of tight junction is abnormal in CLDN14M18V, and CLDN14W56S MDCK cell line in transepithelial electrical resistance (TER) results. Curcumin inhibits a calcium pump (called sarcoplasmic reticulum Ca-ATPase) in the endoplasmic reticulum and decreases proteasomal degradation. In staining and compartmental protein extraction results, some part of accumulated CLDN14M18V protein was transferred to plasma membrane during treating with curcumin. Moreover, the barrier function of tight junction was rescued in CLDN14M18V cell line after treating with curcumin. A part of accumulated CLDN14M18V protein was also transferred to plasma membrane after treating with proteasome inhibitor, MG132, similar to curcumin treated. The CLDN14M18V expression rate in plasma membrane was increased about 7.76 fold compare to MG132 non-treated results. Base on these results, we hope this study could help us to learn more information of the functions, mechanisms or treatments in mutations or deletions of CLAUDIN 14 (CLDN14) gene.

Vor 2 Jahren wurden erstmals die wahrscheinlich wichtigsten Tight Junction Proteine, Claudin-1 und -2 in der Maus beschrieben. An Hand von Sequenzhomologien konnten bis heute dieser 4 Transmembrandomänen Proteinfamilie 18 Mitglieder mit unterschiedlichen Gewebeverteilungen zugeordnet werden. Parallel zum murinen Claudin-1 wurde von (Swisshelm et al. 1999) das humane Claudin-1 mit einer 91 prozentigen Sequenzhomologie zum murinen Protein isoliert und molekulargenetisch beschrieben. In der überwiegenden Mehrzahl von Brusttumorzellinien ist die Expression von hu-CLDN1 verloren gegangen. In der vorliegenden Arbeit sollte deshalb die biologische Funktion des humanen Claudin-1 (hu-CLDN1) und dessen Relevanz während der Tumorgenese untersucht werden. Für diese Aufgabenstellung mußten monoklonale Antikörper mittels DNA Vakzinierung und hu-CLDN1 Retroviren zur effektiven Transduktion von Tumorzellen entwickelt werden. Die Antikörper wurden in ELISA, kompetitiven hu-CLDN1 Peptid ELISA und Western Blot auf ihre hu-CLDN1 Spezifität und Epitoperkennungsstelle überprüft. Für biologische Untersuchungen wurden für die neuen Antikörper optimale immunzytochemische und immunhistochemische Methoden etabliert. Mit Hilfe der monoklonalen Antikörper wurde die zelluläre Proteinexpression und die Lokalisation, als auch die Expression von hu-CLDN1 in Gewebeschnitten von Tumoren- und Normalgewebe untersucht. Vor 2 Jahren wurden erstmals die wahrscheinlich wichtigsten Tight Junction Proteine, Claudin-1 und -2 in der Maus beschrieben. An Hand von Sequenzhomologien konnten bis heute dieser 4 Transmembrandomänen Proteinfamilie 18 Mitglieder mit unterschiedlichen Gewebeverteilungen zugeordnet werden. Parallel zum murinen Claudin-1 wurde von (Swisshelm et al. 1999) das humane Claudin-1 mit einer 91 prozentigen Sequenzhomologie zum murinen Protein isoliert und molekulargenetisch beschrieben. In der überwiegenden Mehrzahl von Brusttumorzellinien ist die Expression von hu-CLDN1 verloren gegangen. In der vorliegenden Arbeit sollte deshalb die biologische Funktion des humanen Claudin-1 (hu-CLDN1) und dessen Relevanz während der Tumorgenese untersucht werden. Für diese Aufgabenstellung mußten monoklonale Antikörper mittels DNA Vakzinierung und hu-CLDN1 Retroviren zur effektiven Transduktion von Tumorzellen entwickelt werden. Die Antikörper wurden in ELISA, kompetitiven hu-CLDN1 Peptid ELISA und Western Blot auf ihre hu-CLDN1 Spezifität und Epitoperkennungsstelle überprüft. Für biologische Untersuchungen wurden für die neuen Antikörper optimale immunzytochemische und immunhistochemische Methoden etabliert. Mit Hilfe der monoklonalen Antikörper wurde die zelluläre Proteinexpression und die Lokalisation, als auch die Expression von hu-CLDN1 in Gewebeschnitten von Tumoren- und Normalgewebe untersucht. Für die Reexpression von hu-CLDN1 in Brusttumorzellen wurden retrovirale Genshuttlesysteme hergestellt. Die retroviralen Genshuttlesysteme basieren auf dem MoMuLV Grundgerüst und als Besonderheit wurde der l-NGFR Rezeptor zur schnellen und sicheren Identifkation transduzierter Zellen einkloniert. Hergestellt wurde ein Mock Kontrollvektor (nur l-NGFR) und der hu-CLDN1 Vektor mit l-NGFR. Die hu-CLDN1 retroviralen Überstände wurden verwendet, um verschiedene Brusttumorzellinien zu transduzieren. Die Expression von hu-CLDN1 wurde mittels eigens entwickelter quantitativer gekoppelter Reverser Transkription Polymerase Ketten Reaktion (qRT PCR) in verschiedenen Brusttumorzellinien untersucht. Wie aus der vorliegenden Arbeit ersichtlich, konnten erstmalig monoklonale Antikörper gegen hu-CLDN1 entwickelt werden. Diese sind spezifisch und haben eine hohe Sensitivität gegenüber hu-CLDN1. Desweiteren gelang es erstmals Antikörper gegen alle 4 extra- und intrazellulären Domänen zu gewinnen. Mit diesen Antikörpern gelang es nachzuweisen, daß es sich bei hu-CLDN1 um ein ausschließlich membranständiges Protein handelt. Die Expression des Proteins findet sich nur in konfluenten Zellkulturen von natürlicherweise hu-CLDN1 exprimierenden Brusttumorzellen (T47D, MCF7), wobei die Lokalisation ausschließlich auf die Zell-Zell Kontaktstelle beschränkt ist. Bei der hu-CLDN1 Transduktion in hu-CLDN1 negativen Brusttumorzellinen zeigte sich eine konstitutive mRNA Expression in subkonfluenten und konfluenten Zellkulturen. Allerdings ist fluoreszenzmikroskopisch hu-CLDN1 Protein nur in konfluenten Zellkulturen nachweisbar, wobei diese Tumorzellen das Protein korrekt nur an der Zell-Zell Kontaktstelle einbauen. Offensichtlich haben die hu-CLDN1 negativen Brusttumorzellen noch den intakten Signalweg zur korrekten Expression mit einer noch unbekannten posttranskriptionellen Kontrolle. Bemerkenswert ist in diesem Zusammenhang, daß in Brusttumorzellen, die weder Occludin noch ZO-1 exprimieren (MDA-MB-435) die physiologisch korrekte Expression von hu-CLDN1 existiert. Offensichtlich ist die Lokalisation von hu-CLDN1 von beiden Proteinen unabhängig. Die Analyse von unterschiedlichen hu-CLDN1 positiven und negativen Brusttumorzellen zeigte, daß der Verlust der hu-CLDN1 Expression besser zur in vitro Invasivität von Brusttumorzellen korreliert als der Expressionsverlust von Occludin und ZO-1. Die klinische Relevanz des Verlustes von hu-CLDN1 während der Tumorgenese wurde bei den Expressionsanalysen auf normalen Brust- und Darmgewebe gegenüber transformierten Brust- und Darmgewebe untersucht. Bei der Analyse von normalem Brustgewebe konnte festgestellt werden, daß hu-CLDN1 ein rein epthelial/endotheliales Protein mit eindeutiger Membranlokalisation darstellt. In den untersuchten transformierten Geweben zeigte keines der transformierten Gewebe mehr die membranständige Färbung, sondern nur noch zytoplasmatische Färbung. Desweiteren war eine signifikant reduzierte oder nicht vorhandene hu-CLDN1 Färbung in einer größeren Anzahl von Tumoren zu beobachten. Diese Ergebnisse zeigen, daß der Verlust der Expression zumindest bei der Brust- und Darmtumorentwicklung offensichtlich mit der in vivo Tumorprogression korreliert. Um die Bedeutung der hu-CLDN1 Relokalisation bzw. verringerten Expression während der Tumorgenese zellphysiologisch zu verstehen, wurden in vitro Zellkulturstudien mit hu-CLDN1 negativen Zellinien und ihren hu-CLDN1 transduzierten Tochterpopulationen durchgeführt. In den adhärenten Zellkulturen hat die hu-CLDN1 Expression keinen Einfluß auf Zellwachstum und Apoptose. Allerdings zeigte sich in drei dimensional wachsenden Tumorzellaggregaten, daß die Reexpression von hu-CLDN1 zu einer drastischen Erhöhung der Apoptose führt. Die parazellulären Fluxstudien ergaben, daß bei der Reexpression von hu-CLDN1 in Brusttumorzellen der parazelluläre Flux zwischen den Zellen deutlich zurückgeht. Somit könnte die reduzierte Wachstumskapazität bzw. erhöhte Apoptose in 3 D Kulturen mit einer reduzierten Zugänglichkeit von Nährstoffen und Wachstumsfaktoren in hu-CLDN1 transduzierten Zellen verursacht sein. Dies würde auch erklären, warum bei den untersuchten transformierten noch hu-CLDN1 positiven Brust- und Darmtumorgeweben sich ausschließlich eine zytoplasmatische Lokalisation in den Tumorzellen findet. Während in Organen und Drüsengeweben Epithelien einschichtig vorkommen und die Versorgung der Zellen mit Wachstumsfaktoren basolateral oder apikal durch Diffusion und Mikro-/Makropinozytose erfolgen kann, wachsen Tumorepithelzellen mehrschichtig. Wären die Tight Junctions im Tumor noch intakt, so könnte es zu einer Mangelversorgung und somit zur Apoptose kommen. Für das in vivo Wachstum eines Tumors ist es also notwendig, Membranproteine, die den parazellulären Flux inhibieren, zu verringern. Wie die zellphysiologischen in vitro Studien der vorliegenden Arbeit zeigen, ist es aber möglich, einen tumorinhibierenden Effekt allein durch die Reexpression von hu-CLDN1 unabhängig von Occludin und ZO-1 zu erreichen. In diesem Sinne kann zumindest zellphysiologisch hu-CLDN1 als Tumorsuppressorprotein betrachtet werden
Two years ago probably the most important tight junction proteins in mice were identified: claudin-1 and -2 . By sequence homology up to 18 members could be assigned to this 4 transmembrane protein family. The human claudin-1 with a 91 per cent sequence similarity to the murine claudin-1 was isolated in parallel (Swisshelm et al. 1999) and it has been shown that most breast cancer cell lines lost the expression of hu-CLDN1. The goal of this Ph. D. thesis was the evaluation of the cell physiological function of the human Claudin-1 (hu-CLDN1) and its relevance during tumorigenesis. To investigate the protein expression and cellular homing monoclonal antibodies using DNA vaccination were generated. The antibodies were analyzed for hu-CLDN1 specifity by Western blot and the epitope binding sites were identified by a competetive hu-CLDN1 peptide ELISA. Using the monoclonal antibodies optimal immunocytochemical and immunohistochemical methods for biological methods were established. With these antibodies the cellular expression and localization, and the expression of hu-CLDN1 in tissue slices of tumor versus normal tissues was analyzed. For the reexpression of hu-CLDN1 in breast tumor cells, retroviral shuttle systems were generated, based on a MoMuLV backbone using the l-NGFR receptor for efficient and rapid transduction of breast cancer cells with hu-CLDN1. For this purpose a mock vector (containing l-NGFR only ) and a hu-CLDN1 vector with l-NGFR were cloned. The expression of hu-CLDN1 in various breast cancer cell lines was analysed using quantitative reverse transcription polymerase chain reaction (qRT PCR). The monoclonal antibodies against hu-CLDN1 are specific against hu-CLDN1. In addition antibodies for all 4 extra- and cytosolic domains were generated. Using these antibodies it was shown by immunofluorescence microscopic analysis that hu-CLDN1 is an exclusively membrane located protein. The expression of the protein is detectable only in confluent cell cultures of naturally hu-CLDN1 expressing cell lines (T47-D, MCF7) and only in the areas of direct cell cell contact. The transduction of hu-CLDN1 negative cell lines analyzed by qRT PCR displayed a mRNA expression in subconfluent and confluent cell cultures, while the protein was only detectable in confluent cell cultures. This indicates that the hu-CLDN1 negative breast tumor cell lines still have the intact signal transduction pathway for the correct expression with an up to now unknown posttranscriptional control mechanism of protein expression. In this context it is noteworthy that even occludin and ZO-1 negative breast tumor cell lines (e. g. MDA-MB-435) still have the physiological homing mechanism of hu-CLDN1. Therefore it can be suggested that expression and homing at hu-CLDN1 might be independent of occludin and ZO-1. The analysis of different hu-CLDN1 positive and negative breast tumor cells showed that the loss of expression of hu-CLDN1 correlates more significantly to the tumorigenesis of cells in comparison to occludin and ZO-1. The clinical relevance of the downregulation and loss of hu-CLDN1 was analyzed using expression analysis of breast and colon tissue versus tumor tissue on tissue microarrays. Normal breast tissue displayed a epithelial/endothelial hu-CLDN1 membrane localization only. None of the breast tumor tissue displayed a membrane localization of the hu-CLDN1 however a relocalization to the cytoplasm was evident. Additionally a significant reduction or loss of expression could be detected in the majority of tumor tissues. These results show that the loss of expression of hu-CLDN1 in breast and colon tumors correlates with tumor progression. In order to analyze the relevance of hu-CLDN1 relocalization and reduction of expression during tumorigenesis on a cellphysiological level, in vitro cell culture studies were performed using hu-CLDN1 negative cell lines (MDA-MB-361) and their hu-CLDN1 transduced counterparts. Hu-CLDN1 transduced cells growing in 2 D cell cultures displayed no altered growth or increased levels of apoptosis. However in three dimensional growing tumor cell aggregates the reexpression of hu-CLDN1 results in a significantly increased induction of apoptosis. In addition paracellular flux analysis revealed that reexpression of hu-CLDN1 decreased the paracellular flux significantly. This data suggests that the reduced growth and increased apoptosis in hu-CLDN1 positive tumor cell aggregates could be due to reduced accessibility of growth factors in hu-CLDN1 transduced cell aggregates. This would explain the necessity for the cytoplasmic homing and loss of expression of hu-CLDN1 during tumorprogression in breast and colon tumors. Most epithelia exist as single layers in organs and lobular structures resulting in a good accessibility of growth factors via diffusion or micro-/macropinocytosis. However, carcinoma tumor cells grow in multilayers. Intact tight junction complexes in carcinoma multi layers would result in a reduced accessibility of growth factors and nutrients. Therefore it is suggested that it is essential for the in vivo growth of a tumor to decrease the expression of tight junctions regulating the paracellular flux. According to the molecular and cell physiological studies presented it might be possible to achieve a tumor inhibiting effect by re-expression of hu-CLDN1. Therefore at least from a cell physiological point of view hu-CLDN1 can be considered to be a tumor suppressor protein