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Abstract Background Squamous cell carcinoma of the head and neck (SCCHN) are highly invasive tumours with frequent local and distant recurrence. Metastasis formation requires degradation of the extracellular matrix, which is fulfilled by membrane-associated proteases such as the urokinase plasminogen activator (uPA). WX-UK1 is a competitive active site inhibitor of the protease function of uPA that impairs on the capacity of tumour cells to invade in vitro. Methods In the present study, effects of combinations of WX-UK1 with matrix metalloprotease inhibitors (MMP, galardin®) and cyclooxygenase-2 (COX-2, celecoxib®) inhibitors on tumour cell proliferation, invasion, and angiogenesis induction were evaluated. Matrigel invasion chambers and a spheroid co-cultivation model with human fibroblast served to determine the invasive potential of both FaDu (SCCHN) and HeLa (cervical carcinoma) cells, each treated with combinations of Celecoxib®, Galardin®, and WX-UK1. Results Blocking of single protease systems resulted in a significant 50% reduction of tumour cell invasion using WX-UK1, while the triple combination was even more effective with 80% reduction of invasion. Additionally, a sprouting assay with HUVEC was used to test the anti-angiogenetic potential of the triple combination, resulting in a 40% decrease in the sprouting rate. Conclusions A combined approach targeting different families of proteases and cyclooxygenases represents a promising adjuvant therapy.

Abstract Background Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is frequently over-expressed in simple epithelia, progenitors, embryonic and tissue stem cells, carcinoma and cancer-initiating cells. Besides functioning as a homophilic adhesion protein, EpCAM is an oncogenic receptor that requires regulated intramembrane proteolysis for activation of its signal transduction capacity. Upon cleavage, the extracellular domain EpEX is released as a soluble ligand while the intracellular domain EpICD translocates into the cytoplasm and eventually into the nucleus in combination with four-and-a-half LIM domains protein 2 (FHL2) and β-catenin, and drives cell proliferation. Methods EpCAM cleavage, induction of the target genes, and transmission of proliferation signals were investigated under varying density conditions using confocal laser scanning microscopy, immunoblotting, cell counting, and conditional cell systems. Results EpCAM cleavage, induction of the target genes, and transmission of proliferation signals were dependent on adequate cell-to-cell contact. If cell-to-cell contact was prohibited EpCAM did not provide growth advantages. If cells were allowed to undergo contact to each other, EpCAM transmitted proliferation signals based on signal transduction-related cleavage processes. Accordingly, the pre-cleaved version EpICD was not dependent on cell-to-cell contact in order to induce c-myc and cell proliferation, but necessitated nuclear translocation. For the case of contact-inhibited cells, although cleavage of EpCAM occurred, nuclear translocation of EpICD was reduced, as were EpCAM effects. Conclusion Activation of EpCAM's cleavage and oncogenic capacity is dependent on cellular interaction (juxtacrine) to provide for initial signals of regulated intramembrane proteolysis, which then support signalling via soluble EpEX (paracrine).

Abstract Background The intermediate filament forming protein keratin 8 (K8) is a tumour-associated antigen, which was shown to be over-expressed in a variety of malignancies. Here, we present a study of K8 expression in squamous epithelia of the head and neck area, including normal mucosa, hyperplastic and dysplastic leukoplakia, carcinomas of different sub-localisations, and lymph node metastases. Methods K8 expression was assessed upon immunohistochemistry with specific antibodies in cryosections of primary tumours of the head and neck area. Results K8 expression was characteristic of transformed tissue and marked early stages of disease, i.e. dysplastic oral leukoplakia, but not normal or hyperplastic epithelium. With the exception of carcinomas of the larynx and the tongue, K8 expression also strictly differentiated carcinomas from normal epithelium of the same origin. Furthermore, K8high was characteristic of cells, which had detached from the sites of primary tumours and had been invading the surrounding tissue at the time point of surgery. Conclusion K8 is an excellent marker for head and neck malignancies, which allows for early detection as well as for visualisation of potentially disseminating tumour cells in vivo.

Intraoperative detection of residual malignant cells at tumor margins following excision of primary tumors could help improving surgery and thus patients' outcome. The feasibility of the tumor antigens epidermal growth factor receptor (EGF-R) and epithelial cell adhesion molecule (EpCAM) for antibody-dependent confocal laser scanning endomicroscopy (CLE)-mediated visualization of malignant cells was addressed. Both tumor antigens are highly and frequently expressed in the majority of carcinomas, including head and neck squamous cell carcinomas (HNSCC), and represent prognostic and therapeutic tumor target molecules. FITC-conjugated EGF-R- and EpCAM-specific antibodies served as molecular tools for the detection of antigen-positive cells using the CLE technology. Specificity of both antibodies and their ability to discriminate tumor from non-tumor cells were assessed in vitro with human fibroblasts and PCI-1 HNSCC cell lines, and ex vivo on primary HNSCC samples ( n = 11) and healthy mucosa ( n = 5). Antigen specificity of the used EpCAM-specific antibody was superior to that of the EGF-R-specific antibody both in vitro and ex vivo (100% vs. 31.25%), and allowed visualization of cellular structures in CLE measurements. These results hold promise for possible future applications in humans. [ABSTRACT FROM AUTHOR]

Background: To evaluate the in vivo response by detecting the anti-angiogenic and invasion-inhibiting effects of a triple-combination-therapy in an experimental-small-animal-squamous-cell-carcinoma-model using the "flash-replenishment" (FR) method to assess tissue hemodynamics via contrast-enhanced-ultrasound (CEUS). Methods: Human hypopharynx-carcinoma-cells were subcutaneously injected into the left flank of 22-female-athymic-nude-rats. After seven days of subcutaneous tumor growth, FR-measurements were performed on each rat. Treatment-group and control-group were treated every day for a period of one week, with the treatment-group receiving solvents containing a triple therapy of Upamostat®, Celecoxib® and Ilomastat® and the control-group solvents only. On day seven, follow-up measurements were performed using the same measurement protocol to assess the effects of the triple therapy. VueBox® was used to quantify the kinetic parameters and additional immunohistochemistry analyses were performed for comparison with and validation of the CEUS results against established methods (Proliferation/Ki-67, vascularization/CD31, apoptosis/caspase3). Results: Compared to the control-group, the treatment-group that received the triple-therapy resulted in a reduction of tumor growth by 48.6% in size. Likewise, the immunohistochemistry results showed significant decreases in tumor proliferation and vascularization in the treatment-group in comparison to the control-group of 26%(p≤0.05) and 32.2%(p≤0.05) respectively. Correspondingly, between the baseline and follow-up measurements, the therapy-group was associated with a significant(p ≤ 0.01) decrease in the relative-Blood-Volume(rBV) in both the whole tumor(wt) and hypervascular tumor(ht) areas (p≤0.01), while the control-group was associated with a significant (p≤0.01) increase of the rBV in the wt area and a non-significant increase (p≤0.16) in the ht area. The mean-transit-time (mTT) of the wt and the ht areas showed a significant increase (p≤0.01) in the follow-up measurements in the therapy group. Conclusion: The triple-therapy is feasible and effective in reducing both tumor growth and vascularization. In particular, compared with the placebo-group, the triple-therapy-group resulted in a reduction in tumor growth of 48.6% in size when assessed by CEUS and a significant reduction in the number of vessels in the tumor of 32% as assessed by immunohistochemistry. As the immunohistochemistry supports the CEUS findings, CEUS using the "flash replenishment"(FR) method appears to provide a useful assessment of the anti-angiogenic and invasion-inhibiting effects of a triple combination therapy. [ABSTRACT FROM AUTHOR]

Background: Novel anti-angiogenic treatments are increasingly complementing established cancer therapy strategies in head and neck tumors. Contrast-enhanced magnetic resonance imaging (MRI) can be applied for early and non-invasive therapy monitoring by non-invasive quantitative assessment of tumor microcirculation as in vivo imaging biomarkers of therapy response.Purpose: To monitor the anti-angiogenic effects of a novel combination therapy on experimental head and neck squamous cell carcinomas (HNSCC) with dynamic contrast-enhanced (DCE)-MRI.Material and Methods: Athymic rats (n = 18) with subcutaneous HNSCC xenografts were investigated by DCE-MRI before and after 7 days of a daily triple therapy regimen combining the COX-II-inhibitor celecoxib, the matrix-metalloproteinase-inhibitor GM6001, and the uPA-inhibitor upamostat. Quantitative measurements of tumor blood flow (tBF), tumor blood volume (tBV), and permeability-surface area product (PS) were calculated and validated by immunohistochemistry.Results: Mean tBF and tBV in triple-therapy animals decreased significantly from day 0 to day 7 (tBF, 41.0 ± 14.2 to 20.4 ± 5.7 mL/100 mL/min; P < 0.01; tBV, 17.7 ± 3.9 to 7.5 ± 3.3%; P < 0.01). No significant effects on PS were observed in either group (P > 0.05). Immunohistochemical analysis showed a significantly lower tumor vascularity in the therapy group than in the control group (CD31), significantly fewer Ki-67+ proliferating tumor cells and significantly more Capase-3+ apoptotic tumor cells (P < 0.05). Significant (P < 0.05) correlations were observed between tBF/tBV and CD31 (tBF, r = 0.84; tBV, r = 0.70), tBV and Ki-67 (r = 0.62), as well as tBF and caspase-3 (r = -0.64).Conclusion: DCE-MRI may be a suitable tool for the non-invasive monitoring of the anti-vascular effects of this innovative triple therapy regimen with potential for clinical translation. [ABSTRACT FROM AUTHOR]

Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is highly and frequently expressed in carcinomas and (cancer-)stem cells, and which plays an important role in the regulation of stem cell pluripotency. We show here that murine EpCAM (mEpCAM) is subject to regulated intramembrane proteolysis in various cells including embryonic stem cells and teratocarcinomas. As shown with ectopically expressed EpCAM variants, cleavages occur at α-, β-, γ-, and ε-sites to generate soluble ectodomains, soluble Aβ-like-, and intracellular fragments termed mEpEX, mEp-β, and mEpICD, respectively. Proteolytic sites in the extracellular part of mEpCAM were mapped using mass spectrometry and represent cleavages at the α- and β-sites by metalloproteases and the b-secretase BACE1, respectively. Resulting C-terminal fragments (CTF) are further processed to soluble Aβ-like fragments mEp-β and cytoplasmic mEpICD variants by the g-secretase complex. Noteworthy, cytoplasmic mEpICD fragments were subject to efficient degradation in a proteasome-dependent manner. In addition the γ-secretase complex dependent cleavage of EpCAM CTF liberates different EpICDs with different stabilities towards proteasomal degradation. Generation of CTF and EpICD fragments and the degradation of hEpICD via the proteasome were similarly demonstrated for the human EpCAM ortholog. Additional EpCAM orthologs have been unequivocally identified in silico in 52 species. Sequence comparisons across species disclosed highest homology of BACE1 cleavage sites and in presenilin-dependent γ-cleavage sites, whereas strongest heterogeneity was observed in metalloprotease cleavage sites. In summary, EpCAM is a highly conserved protein present in fishes, amphibians, reptiles, birds, marsupials, and placental mammals, and is subject to shedding, γ-secretase-dependent regulated intramembrane proteolysis, and proteasome-mediated degradation. [ABSTRACT FROM AUTHOR]

In many solid tumors, cancer stem cells (CSC) represent a population with tumor-initiating, self-renewal, and differentiation potential, which can be identified by surface protein markers. No generally applicable markers are yet known for renal cell carcinoma (RCC). Two RCC cell lines (RCC-26, RCC-53) were found to differ widely in their capacity to form spheres in vitro and to establish tumors in mice, potentially reflecting differences in CSC content. A subpopulation expressing the CXC chemokine receptor 4 (CXCR4) was present only in the more tumorigenic cell line RCC-53. When grown as spheres, most of the RCC-53 cells were CXCR4-positive, expressed stem cell-associated transcription factor genes at elevated levels, and were more resistant toward the tyrosine kinase inhibitors sunitinib, sorafenib, and pazopanib. Sorted CXCR4-positive cells exhibited greater capacity for sphere formation and tumor growth-inducing potential in vivo than CXCR4-negative cells. Significantly, higher CXCR4 mRNA levels in primary RCC tumors from patients with localized but not disseminated disease predicted shorter survival. Downregulation of CXCR4 expression by small interfering RNA (siRNA) or pharmacological inhibition by AMD3100 compromised tumor sphere formation, viability of CXCR4-positive cells, and increased their responsiveness toward tyrosine kinase inhibitors. In conclusion, CXCR4 identifies a subpopulation of tumor-initiating cells in RCC cell lines and plays a role in their maintenance. The relative insensitivity of such cells to tyrosine kinase inhibitors might contribute to the development of therapy resistance in RCC patients. Future therapies therefore could combine blockade of the CXCR4 signaling pathway with standard therapies for more effective treatments of metastatic RCC. S TEM C ells 2013;31:1467-1476 [ABSTRACT FROM AUTHOR]

BACKGROUND: The pentaspan protein CD133 (Prominin-1) is a predictive marker and part of the signature of tumour-initiating cells (TICs) for various cancer entities. METHODS: The correlation of CD133 expression with clinical parameters was assessed in primary samples of head and neck squamous cell carcinomas (n=98) and normal mucosas (n=24). RESULTS: A gradual and inversely proportional correlation between CD133 expression in primary tumours and decreased overall survival was observed, along with a positive correlation with the presence of lymph node metastases. CONCLUSIONS: CD133 has the potential of being a novel clinically relevant prognostic marker for head and neck malignancies, which is possibly involved in regulation of tumourigenicity. [ABSTRACT FROM AUTHOR]

The study of tumourigenesis commonly involves the use of established cell lines or single cell suspensions of primary tumours. Standard methods for the generation of short-term tumour cell cultures include the disintegration of tissue based on enzymatic and mechanical stress. Here, we describe a simple and rapid method for the preparation of single cells from primary carcinomas, which is independent of enzymatic treatment and feeder cells. Tumour biopsies are processed to 1 mm³ cubes termed explants, which are cultured 1--3 days on agarose-coated well plates in specified medium. Through incisions generated in the explants, single cells are retrieved and collected from the culture supernatant and can be used for further analysis including in vitro and in vivo studies. Collected cells retain tumour-forming capacity in xenotransplantation assays, mimic the phenotype of the primary tumour, and facilitate the generation of cell lines. [ABSTRACT FROM AUTHOR]

Abstract: Transketolase-like protein 1 (TKTL1) is a member of the family of transketolase enzymes of which the founder member transketolase (TKT) is known to play a central role in the non-oxidative part of the pentose phosphate pathway. According to several publications TKTL1 is the only family member, whose expression is substantially de-regulated in a variety of solid tumours. Over-expression of TKTL1 correlates with poor prognosis of cancer patients and TKTL1 itself represents a potential therapeutic target owing to its possible involvement in the regulation of the proliferation and metabolism of cancer cells. We show that exogenously expressed TKTL1 provides HEK293 cells with moderate growth advantages under standard culture conditions, while protecting cells from growth factor withdrawal-induced apoptosis. Importantly, we identified TKTL1 with the JFC12T10 antibody as a 65kDa protein, which was however absent in most tumour cell lines tested. Primary head and neck squamous cell carcinomas of various localisations were characterised by a focal pattern with single cells strongly expressing TKTL1, rather than by a homogeneous expression pattern within the tumour mass. [ABSTRACT FROM AUTHOR]

Background: Squamous cell carcinoma of the head and neck (SCCHN) are highly invasive tumours with frequent local and distant recurrence. Metastasis formation requires degradation of the extracellular matrix, which is fulfilled by membrane-associated proteases such as the urokinase plasminogen activator (uPA). WX-UK1 is a competitive active site inhibitor of the protease function of uPA that impairs on the capacity of tumour cells to invade in vitro. Methods: In the present study, effects of combinations of WX-UK1 with matrix metalloprotease inhibitors (MMP, galardin®) and cyclooxygenase-2 (COX-2, celecoxib®) inhibitors on tumour cell proliferation, invasion, and angiogenesis induction were evaluated. Matrigel invasion chambers and a spheroid co-cultivation model with human fibroblast served to determine the invasive potential of both FaDu (SCCHN) and HeLa (cervical carcinoma) cells, each treated with combinations of Celecoxib®, Galardin®, and WX-UK1. Results: Blocking of single protease systems resulted in a significant 50% reduction of tumour cell invasion using WX-UK1, while the triple combination was even more effective with 80% reduction of invasion. Additionally, a sprouting assay with HUVEC was used to test the anti-angiogenetic potential of the triple combination, resulting in a 40% decrease in the sprouting rate. Conclusions: A combined approach targeting different families of proteases and cyclooxygenases represents a promising adjuvant therapy. [ABSTRACT FROM AUTHOR]

Abstract: Chronic administration of nicotine is followed by a general stimulation of brain metabolism that results in a distinct increase of glucose transport protein densities for Glut1 and Glu3, and local cerebral glucose utilization (LCGU). This increase of LCGU might be paralleled by an enhanced production of lactate. Therefore, the question arose as to whether chronic nicotine infusion is accompanied by increased local densities of monocarboxylate transporter MCT1 in the brain. Secondly, we inquired whether LCGU might be correlated with local densities of MCT1 during normal conditions and after chronic nicotine infusion. Nicotine was given subcutaneously for 1 week by osmotic mini-pumps and local densities of MCT1 were measured by immunoautoradiographic methods in cryosections of rat brains. MCT1 density was significantly increased in 21 of 32 brain structures investigated (median increase 15.0±3.6%). Immunohistochemical stainings of these substructures revealed an over-expression of MCT1 within endothelial cells and astrocytes of treated animals. A comparison of 23 MCT1 densities with LCGU measured in the same structures in a previous study revealed a partial correlation between both parameters under control conditions and after chronic nicotine infusion. 10 out of 23 brain areas, which showed a significant increase of MCT1 density due to chronic nicotine infusion, also showed a significant increase of LCGU. In summary, our data show that chronic nicotine infusion induces a moderate increase of local and global density of MCT1 in defined brain structures. However, in terms of brain topologies and substructures this phenomenon did partially match with increased LCGU. It is concluded that MCT1 transporters were upregulated during chronic nicotine infusion at the level of brain substructures and, at least partially, independently of LCGU. [Copyright &y& Elsevier]

Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein that is expressed on subsets of normal epithelia, numerous stem- and progenitor-type cells, and most carcinomas and highly overexpressed on cancer-initiating cells. The role of EpCAM in early development, particularly in stem-like cells, has remained unclear. Here, we show that the maintenance of self-renewal in murine embryonic stem (ES) cells depends on the high-level expression of EpCAM. Cultivation of ES cells under differentiation conditions in the absence of leukemia inhibitory factor (LIF) caused down-regulation of EpCAM along with decreased expression of cellular myelocytomatosis oncogene (c-Myc), Sex-determining region Y-Box 2, Octamer 3/4 (Oct3/4), and Stat3. As a consequence ES cells were morphologically differentiated and ceased to proliferate. RNA interference-mediated inhibition of EpCAM expression under self-renewal conditions resulted in quantitatively decreased proliferation, decreased Oct3/4, SSEA-1, and c-Myc expression, and diminished alkaline phosphatase activity. Conversely, exogenous expression of EpCAM partially compensated for the requirement of ES cells for LIF to retain a stem cell phenotype. Thus, murine EpCAM is a transmembrane protein, which is essential but by itself is not sufficient for maintenance of the ES cell phenotype. [ABSTRACT FROM AUTHOR]

EpCAM was found to be overexpressed on epithelial progenitors, carcinomas and cancer-initiating cells. The role of EpCAM in proliferation, and its association with cancer is poorly explained by proposed cell adhesion functions. Here we show that regulated intramembrane proteolysis activates EpCAM as a mitogenic signal transducer in vitro and in vivo. This involves shedding of its ectodomain EpEX and nuclear translocation of its intracellular domain EpICD. Cleavage of EpCAM is sequentially catalysed by TACE and presenilin-2. Pharmacological inhibition or genetic silencing of either protease impairs growth-promoting signalling by EpCAM, which is compensated for by EpICD. Released EpICD associates with FHL2, β-catenin and Lef-1 to form a nuclear complex that contacts DNA at Lef-1 consensus sites, induces gene transcription and is oncogenic in immunodeficient mice. In patients, EpICD was found in nuclei of colon carcinoma but not of normal tissue. Nuclear signalling of EpCAM explains how EpCAM functions in cell proliferation. [ABSTRACT FROM AUTHOR]

Background: CD44 splice variants are long-known as being associated with cell transformation. Recently, the standard form of CD44 (CD44s) was shown to be part of the signature of cancer stem cells (CSCs) in colon, breast, and in head and neck squamous cell carcinomas (HNSCC). This is somewhat in contradiction to previous reports on the expression of CD44s in HNSCC. The aim of the present study was to clarify the actual pattern of CD44 expression in head and neck epithelia. Methods: Expression of CD44s and CD44v6 was analysed by immunohistochemistry with specific antibodies in primary head and neck tissues. Scoring of all specimens followed a two-parameters system, which implemented percentages of positive cells and staining intensities from - to +++ (score =%6intensity; resulting max. score 300). In addition, cell surface expression of CD44s and CD44v6 was assessed in lymphocytes and HNSCC. Results: In normal epithelia CD44s and CD44v6 were expressed in 60-95% and 50-80% of cells and yielded mean scores with a standard error of a mean (SEM) of 249.5±14.5 and 198±11.13, respectively. In oral leukoplakia and in moderately differentiated carcinomas CD44s and CD44v6 levels were slightly increased (278.9±7.16 and 242±11.7; 291.8±5.88 and 287.3±6.88). Carcinomas in situ displayed unchanged levels of both proteins whereas poorly differentiated carcinomas consistently expressed diminished CD44s and CD44v6 levels. Lymphocytes and HNSCC lines strongly expressed CD44s but not CD44v6. Conclusion: CD44s and CD44v6 expression does not distinguish normal from benign or malignant epithelia of the head and neck. CD44s and CD44v6 were abundantly present in the great majority of cells in head and neck tissues, including carcinomas. Hence, the value of CD44s as a marker for the definition of a small subset of cells (i.e. less than 10%) representing head and neck cancer stem cells may need revision. [ABSTRACT FROM AUTHOR]

Background: The intermediate filament forming protein keratin 8 (K8) is a tumour-associated antigen, which was shown to be over-expressed in a variety of malignancies. Here, we present a study of K8 expression in squamous epithelia of the head and neck area, including normal mucosa, hyperplastic and dysplastic leukoplakia, carcinomas of different sub-localisations, and lymph node metastases. Methods: K8 expression was assessed upon immunohistochemistry with specific antibodies in cryosections of primary tumours of the head and neck area. Results: K8 expression was characteristic of transformed tissue and marked early stages of disease, i.e. dysplastic oral leukoplakia, but not normal or hyperplastic epithelium. With the exception of carcinomas of the larynx and the tongue, K8 expression also strictly differentiated carcinomas from normal epithelium of the same origin. Furthermore, K8high was characteristic of cells, which had detached from the sites of primary tumours and had been invading the surrounding tissue at the time point of surgery. Conclusion: K8 is an excellent marker for head and neck malignancies, which allows for early detection as well as for visualisation of potentially disseminating tumour cells in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Screening of head and neck carcinoma patients with the proteomics-based AMIDA technology yielded a set of tumour-associated antigens, including the intermediate filament protein cytokeratin 8 (CK8). The expression pattern and specificity of CK8 was compared with those of the established markers pan-cytokeratins and CK13, and with that of the proliferation marker Ki67. Expression of CK8 correlated positively with malignancies of the head and neck areas. CK8 was not expressed in healthy epithelium, except for some rare cases of cells of the basal layer and laryngeal tissue. In contrast, the vast majority of head and neck squamous cell carcinomas and metastases strongly expressed CK8. Interestingly, CK8 de novo expression correlated with dysplastic areas of oral leukoplakic lesions, while hyperplastic leukoplakia remained CK8-negative but strongly panCK and CK13 positive. Thus, CK8 is an attractive marker molecule for a differentiated diagnosis of leukoplakia and head and neck carcinomas, which possesses notedly improved specificity as compared with panCK and CK13. [Copyright &y& Elsevier]

The overall survival rate of patients suffering from carcinomas has remained poor and nearly unchanged over the last decades. This is mainly due to the so-called minimal residual disease, i.e., remaining tumor cells that overcome surgery and/or radiotherapy and are the cause of locoregional and distant metastases. To metastasize, tumor cells take advantage of proteases to invade and remodel surrounding tissues. Here, we analyzed the efficiency of WX-UK1, a novel 3-amidinophenylalanine-based inhibitor of the uPA system, at inhibiting the invasive capacity of carcinoma cells. First, uPAR expression was characterized in different carcinoma cell lines, including SCCHN, breast and cervical carcinoma. Thereafter, the invasive potential of these cell lines was determined using Matrigel invasion chambers and a spheroid cocultivation model with human fibroblasts. uPAR expression levels correlated positively with invasion capacity, which could be significantly inhibited by WX-UK1. A decrease of tumor cell invasion by up to 50% was achieved in both models with the SCCHN line FaDu and the cervical carcinoma line HeLa after treatment with WX-UK1. Thus, our results demonstrate the potential of WX-UK1 in vitro as a promising adjuvant antimetastatic therapy of carcinomas. © 2004 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

One hundred patients with symptomatic peripheral vascular disease were treated with a directional atherectomy catheter; 153 lesions comprising 98 stenoses and 55 occlusions were located in the iliac (n=22), superficial femoral (n = 114), popliteal (n=16), and anterior tibial (n=1) arteries. The majority of these patients were poor candidates for balloon angioplasty because of the complexity of lesions. There were 70 eccentric and 28 concentric lesions and 55 occlusions (mean length 4.2 ± 2.9 cm). Acute success rate was 94% for both stenoses and occlusions. Four patients were treated in both lower extremities. The stenoses were reduced from 85 ± 12% to 12 ± 10% acutely (occlusions 100% to 9 ± 9%). Six-month angiographic follow-ups were performed in 81% of treated patients, the others refusing angiography. Mean stenosis after six months was 33 ± 25% (occlusions 44 ± 28%). Restenosis (> 50%) was found in 20% of treated lesions: 26% in concentric lesions, 8% in eccentric lesions, and 32% in occluded vessels. Treatment of peripheral vascular disease with the atherectomy device is safe and effective therapy with good long-term results. These results were obtained in complex lesions with 55 occlusions. Atherectomy seems to be particularly beneficial in the treatment of eccentric and complex stenoses and is not limited by occlusion or calcification. Furthermore, insight into the pathogenesis of arteriosclerosis and the development of restenosis is enabled by analysis of removed plaque material. [ABSTRACT FROM AUTHOR]

Human epithelial cell adhesion molecule (HEPCAM) is a tumor-associated antigen frequently expressed in carcinomas, which promotes proliferation after regulated intramembrane proteolysis. Here, we describe extracellular shedding of HEPCAM at two α-sites through a disintegrin and metalloprotease (ADAM) and at one β-site through BACE1. Transmembrane cleavage by γ-secretase occurs at three γ-sites to generate extracellular A β-like fragments and at two ε-sites to release human EPCAM intracellular domain HEPICD, which is efficiently degraded by the proteasome. Mapping of cleavage sites onto three-dimensional structures of HEPEX cis-dimer predicted conditional availability of α- and β-sites. Endocytosis of HEP-CAM warrants acidification in cytoplasmic vesicles to dissociate protein cis-dimers required for cleavage by BACE1 at low pH values. Intramembrane cleavage sites are accessible and not part of the structurally important transmembrane helix dimer crossing region. Surprisingly, neither chemical inhibition of cleavage nor cellular knock-out of HEPCAM using CRISPR-Cas9 technology impacted the adhesion of carcinoma cell lines. Hence, a direct function of HEPCAM as an adhesion molecule in carcinoma cells is not supported and appears to be questionable. [ABSTRACT FROM AUTHOR]

B cell-specific gene ablation of Notch2 results in the loss of the marginal zone (MZ) B-cell lineage. To analyze the effects of constitutive Notch2 signaling in B cells, we have generated a transgenic mouse strain that allows the conditional expression of a constitutively active, intracellular form of Notch2 (Notch2IC). Expression of Notch2lC at the earliest developmental stages of the B-cell lineage completely abolished B-cell generation and led to the development of ectopic T cells In the bone marrow (BM), showing that Notch2lC is acting redundantly with NotchllC in driving ectopic T-cell differentiation. In B cells clearly committed to the B-cell lineage induction of Notch2lC drove all cells toward the MZ B-cell compartment at the expense of follicular B cells. Notch2lCexpressing B cells reflected the phenotype of wild-type MZ B cells for their localization in the MZ, the expression of characteristic surface markers, their enhanced proliferation after stimulation, and increased basal activity of Akt, Erk, and Jnk. Notch2lC-driven MZ B-cell generation in the spleen was achieved even in the absence of CD19. Our results implicate that a constitutive Notch2 signal in transitional type 1 B cells is sufficient to drive MZ B-cell differentiation.1) [ABSTRACT FROM AUTHOR]

Abstract: We reported the identification of tumour-associated antigens from head and neck carcinomas, including cytokeratin 8 (CK8). These antigens were isolated based on the humoral immune response they elicit in vivo using the antibody-mediated identification of antigens technology. Unlike healthy squamous epithelium, tumour cells displayed CK8 at the plasma membrane. However, the actual presence of CK8 at the plasma membrane is still a matter of debate. Here, we have analyzed the expression of CK8 in detail using confocal laser scanning microscopy and circumstantiated its localization at the plasma membrane of carcinoma cells. Healthy human tissues were devoid of CK8 at the membrane, with the exception of hepatocytes. Moreover, membrane-associated CK8 molecules experienced a re-distribution throughout mitosis, which was associated with phosphorylation at serine 73. Phosphorylated CK8 redistributed into dense speckles and relocated to the plasma membrane upon cytokinesis. Thus, CK8 possesses genuine extracellular epitopes on tumour cells, which may represent valuable targets for therapy. [Copyright &y& Elsevier]

Recently, we described a new target-identification technology, autoantibody-mediated identification of antigens (AMIDA). AMIDA takes advantage of autologous serum autoantibodies to identify disease-associated antigens. Here, we evaluated the allogenic variant of AMIDA (allo-AMIDA), using permanent cancer cell lines as an antigen-pool rather than primary biopsy samples. Twelve different proteins were retrieved exclusively with antibodies from cancer patients, but not from healthy donors. The expression of three of these antigens, e-FABP, hnRNP H, and Grb2, was evaluated in more detail. All three proteins were strongly overexpressed in primary carcinomas and metastases thereof, as compared to healthy epithelium. Additionally, serum reactivity against e-FABP was detected in 20% of cancer patients but only 2% of healthy volunteers. In summary, we demonstrate that permanent cancer cell lines represent a reliable source for tumour-associated antigens. Moreover, we show that allo-AMIDA is suitable for the identification of tumour-specific antigens overcoming the limitations of autologous screening techniques. [Copyright &y& Elsevier]

The tissue-specific glycosylation of the carcinoma (CA)-associated antigen epithelial cell adhesion molecule (EpCAM) was studied in 60 patients suffering from head and neck CAs, and 26 pairs of autologous healthy thyroid and CA biopsies. EpCAM was glycosylated in all tumor samples in which its expression was detectable (73%). Additionally, in 80.7% of patients, tumor-derived EpCAM was heavily glycosylated while EpCAM derived from autologous thyroid was not (76.2%) or weakly (23.8%). Four cases showed a similar glycosylation pattern (15.3%) and one case displayed a reverse pattern (3.8%). Additionally, the expression and glycosylation of EpCAM were assessed in tumor adjacent and distant tissue. EpCAM was glycosylated in tumor-adjacent while it was not or only weakly expressed in tumor distant tissue where it was unglycosylated. Thus, EpCAM is differentially glycosylated in healthy tissue and tumor cells of the head and neck area. [Copyright &y& Elsevier]

Tumors express specific antigens, which are targets of a humoral immune response. Such specific tumor-associated antigens are of increasing importance for diagnosis and immunotherapy. They allow the detection and targeting of transformed cells in vivo. Here, we describe a new technique for the isolation and identification of tumor antigens that constitute humoral immune responses. The method is an a1utoantibody-m1ediated i1d1entification of a1ntigens (AMIDA) derived from tumor tissue lysates with autologous antibodies, followed by a two-dimensional gel electrophoresis and mass spectrometry analysis. We applied AMIDA to head and neck carcinoma patients and identified 35 antigens, which can be classified in three subgroups: i.) known tumor-associated antigens including SEREX-Ag, ii.) autoimmune disease-associated antigens, and iii.) proteins of unknown function. The expression profile and the validation of a subset of antigens using specific antibodies, the real-time PCR and BioPlex technologies, will be discussed in more detail. Thus, we demonstrate here the potential of AMIDA as a promising tool for the detection of tumor antigens in their native context, i.e. in tumor cells, and describe new protocols for the validation of such proteins. [ABSTRACT FROM AUTHOR]