Biomedical subjects

Reinhard Zeidler

Publications and source records attributed to Reinhard Zeidler.

14 recordsLinked to original sources

PubMed2006

Carcinoma-associated eIF3i overexpression facilitates mTOR-dependent growth transformation.

Molecular processes controlling mRNA translation are complex, multilayered, and their deregulation can lead to cancer pathogenesis. Eukaryotic initiation factor 3 (eIF3) is involved in the initiation process of protein translation and overexpression of its subunit eukaryotic translation initiation factor i (eIF3i) has been observed in carcinomas. Nevertheless, the potential role of eIF3i in carcinogenesis is poorly understood. Here, we show that in vitro overexpression of human eIF3i resulted in cell size increase, proliferation enhancement, cell-cycle progression, and anchorage-independent growth. Without external stimuli, eIF3i overexpressing cells arrested in G1/G0 phase, demonstrating the requirement of additional growth signals. Inhibition of the kinase mTOR, a key player in the integration of nutrition and growth signals into protein synthesis, with rapamycin reduced serine phosphorylation of eIF3i and resulted in a loss of anchorage-independent growth. Thus, eIF3i overexpression fosters the integration of growth signals by mTOR into the mRNA translation process, promoting protein synthesis and tumor growth.

Amino Acid Sequence↗

PubMed2005

Selection of CMV-specific CD8+ and CD4+ T cells by mini-EBV-transformed B cell lines.

Efficient protocols to generate cytomegalovirus (CMV)-specific T cells are required for adoptive immunotherapy. Recombinant Epstein-Barr virus (EBV) vectors called mini-EBV can be used to establish permanent B cell lines in a single step, which present the CMV antigen pp65 in a constitutive manner. These B cell lines, coined pp65 mini-LCL, were successfully used to reactivate and expand CMV-specific cytotoxic T cells. Here we evaluate this pp65 mini-EBV system in closer detail, focusing on (1) the quantification of T cells with specific effector function and (2) the identification of CMV-specific CD4(+) helper T cells. The co-expansion of various functional CMV epitope specificities was demonstrated by IFN-gamma enzyme-linked immunospot assay (ELISPOT) assays and HLA-peptide tetramer staining. Single-cell cloning resulted in both CD4(+) and CD8(+) T cell clones, the majority of which was CMV specific. Thus, mini-LCL present the pp65 antigen on HLA class I and II, mobilizing both arms of the T cell response. Using a peptide library covering the pp65 sequence for further analysis of T cell clones, we identified new pp65 CD8(+) and CD4(+) T cell epitopes.

Adult↗

PubMed2004

The tumour-associated antigen EpCAM upregulates the fatty acid binding protein E-FABP.

The epithelial cell adhesion molecule, EpCAM, is a transmembrane glycoprotein associated with both benign and malignant proliferation. In cancer cells, expression levels of this tumour-associated antigen correlate positively with the grade of dysplasia and are also a negative prognostic factor for breast cancer patients. De novo expression of EpCAM resulted in the rapid upregulation of the proto-oncogene c-Myc along with enhanced cell proliferation and metabolism. Here, we analyzed the effects of EpCAM onto the proteome of epithelial cells. The epidermal fatty acid binding protein, E-FABP, was identified as a new EpCAM-regulated protein. E-FABP is a major target of c-Myc and was rapidly upregulated upon induction of EpCAM. Additionally, E-FABP levels correlated with the amount of EpCAM in permanent squamous cell carcinoma lines and in vivo in primary head and neck carcinomas. Taken together, these results provide further evidence for the direct involvement of EpCAM in signalling processes, gene regulation, and cellular metabolism supporting its important role in tumour biology.

Antigens, Differentiation↗

PubMed2004

Allogenic antibody-mediated identification of head and neck cancer antigens.

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.

Adaptor Proteins, Signal Transducing↗

PubMed2004

The carcinoma-associated antigen EpCAM upregulates c-myc and induces cell proliferation.

Epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein expressed on adenomatous and simple epithelia, where it is involved in homophilic adhesion at the basolateral membrane. Carcinomas strongly overexpress EpCAM through an, as yet, unknown mechanism. Interestingly, otherwise EpCAM-negative squamous epithelia are seen to express EpCAM concomitant with their transformation and de-differentiation. The amount of EpCAM and the number of expressing cells both increase with the grade of dysplasia. Despite an important amount of data correlating the expression of EpCAM with cellular proliferation and de-differentiation, such as the coexpression with Ki-67, a marker for proliferation, it is unknown whether EpCAM may directly contribute to carcinogenesis. Here, we show that EpCAM has a direct impact on cell cycle and proliferation, and the ability to rapidly upregulate the proto-oncogene c-myc and cyclin A/E. Human epithelial 293 cells as well as murine NIH3T3 fibroblasts expressing EpCAM had a decreased requirement for growth factors, enhanced metabolic activity and colony formation capacity. Importantly, the inhibition of EpCAM expression with antisense mRNA led to a strong decrease in proliferation and metabolism in human carcinoma cells. Moreover, domain swapping experiments demonstrated that the intracellular part of EpCAM is necessary and sufficient to transduce the effects described. Thus, the data presented here highlight the role of EpCAM, demonstrating for the first time a direct link to cell cycle and proliferation.

3T3 Cells↗

PubMed2004

Identification of Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) target proteins by proteome analysis: activation of EBNA2 in conditionally immortalized B cells reflects early events after infection of primary B cells by EBV.

The Epstein-Barr virus (EBV) is a ubiquitous B-lymphotropic herpesvirus associated with several malignant tumors, e.g., Burkitt's lymphoma and Hodgkin's disease, and is able to efficiently immortalize primary B lymphocytes in vitro. The growth program of infected B cells is initiated and maintained by the viral transcription factor EBV nuclear antigen 2 (EBNA2), which regulates viral and cellular genes, including the proto-oncogene c-myc. In our study, patterns of protein expression in B cells with and without EBNA2 were analyzed by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. For this purpose, we used a conditional immortalization system for EBV, a B cell line (EREB2-5) that expresses an estrogen receptor-EBNA2 fusion protein. In order to discriminate downstream targets of c-Myc from c-Myc-independent EBNA2 targets, we used an EREB2-5-derived cell line, P493-6, in which c-Myc is expressed under the control of a tetracycline-regulated promoter. Of 20 identified EBNA2 target proteins, 11 were c-Myc dependent and therefore most probably associated with proliferation, and one of these proteins was a posttranslationally modified protein, i.e., hypusinylated eIF5a. Finally, to estimate the relevance of EBNA2 targets during early EBV infection, we analyzed the proteomes of primary B cells before and after infection with EBV. The protein expression pattern induced upon EBV infection was similar to that following EBNA2 activation. These findings underscore the value of EREB2-5 cells as an appropriate model system for the analysis of early events in the process of EBV-mediated B-cell immortalization.

B-Lymphocytes↗

PubMed2003

Tumor-specific glycosylation of the carcinoma-associated epithelial cell adhesion molecule EpCAM in head and neck carcinomas.

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.

Adult↗

PubMed2002

Impaired monocyte function in cancer patients: restoration with a cyclooxygenase-2 inhibitor.

Epidemiological data and animal models have provided evidence that nonsteroidal antiinflammatory drugs (NSAIDs) have an anticancer effect. However, the molecular mechanisms underlying these antineoplastic effects are not well understood. We described previously that expression levels of the chemokine receptor, CCR5, and the beta2-integrin, Mac-1, were down-regulated on primary monocytes after incubation in supernatants from human carcinoma cell lines, and that this down-regulation resulted in impaired monocyte function with respect to migration and adhesion. We now demonstrate that these impairments are also present in vivo. Monocytes from cancer patients displayed significantly reduced CCR5 levels and migration capacities in comparison to cells from healthy donors. Because migration is necessary for the antitumor activity of monocytes/macrophages, these deficits may contribute to the suppressed immune system seen in cancer patients. In a clinical study, we analyzed the effect of a selective COX-2 inhibitor, Rofecoxib, on the migration of monocytes derived from cancer patients. The results revealed significant improvement in migration equal to those levels seen in healthy donors. We conclude that in patients with cancer, the intake of Rofecoxib for 3 wk leads to significant restoration of monocyte function. These data may, at least in part, help explain the anticancer effects of NSAIDs.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2002

B cells immortalized by a mini-Epstein-Barr virus encoding a foreign antigen efficiently reactivate specific cytotoxic T cells.

Lymphoblastoid cell lines (LCLs) are human B cells latently infected and immortalized by Epstein-Barr virus (EBV). Presenting viral antigens, they efficiently induce EBV-specific T-cell responses in vitro. Analogous ways to generate T-cell cultures specific for other antigens of interest are highly desirable. Previously, we constructed a mini-EBV plasmid that consists of less than half the EBV genome, is unable to cause virus production, but still immortalizes B cells in vitro. Mini-EBV-immortalized B-cell lines (mini-LCLs) are efficiently produced by infection of B cells with viruslike particles carrying only mini-EBV DNA. Mini-EBV plasmids can be engineered to express an additional gene in immortalized B cells. Here we present a mini-EBV coding for a potent CD8(+) T-cell antigen, the matrix phosphoprotein pp65 of human cytomegalovirus (CMV). By means of this pp65 mini-EBV, pp65-expressing mini-LCLs could be readily established from healthy donors in a one-step procedure. We used these pp65 mini-LCLs to reactivate and expand effector T cells from autologous peripheral blood cells in vitro. When generated from cytomegalovirus (CMV)-seropositive donors, these effector T-cell cultures displayed strong pp65-specific HLA-restricted cytotoxicity. A large fraction of CD8(+) T cells with pp65 epitope specificity was present in such cultures, as demonstrated by direct staining with HLA/peptide tetramers. We conclude that the pp65 mini-EBV is an attractive tool for CMV-specific adoptive immunotherapy. Mini-EBVs could also facilitate the generation of T cells specific for various other antigens of interest.

Antigens, Viral↗

PubMedDate not supplied

Cloning and characterisation of a 1.1 kb fragment of the carcinoma-associated epithelial cell adhesion molecule promoter.

The epithelial cell adhesion molecule (EpCAM) is a transmembrane protein associated with a variety of carcinomas, where EpCAM is often strongly up-regulated or, as in the case of squamous cell carcinomas, de novo expressed. The molecular mechanisms underlying the transcriptional regulation of EpCAM are poorly understood. So far, a 570 bp fragment has been cloned and shown to have specific transcriptional activity, which was negatively-regulated upon the induction of the transcription factor NF-kappa B. In the present study we have cloned a 1100 bp fragment of the EpCAM promoter containing the 570 bp fragment and additional 550 bp upstream. We demonstrate that both fragments have strong synergistic effects with respect to transcriptional activity in EpCAM-positive cells. Furthermore, the 1100 bp fragment was likewise negatively-regulated upon TNF alpha and IFN alpha treatment, thus retaining silencer sequences.

Antigens, Neoplasm↗

PubMedDate not supplied

Antitumour effects of a bispecific trivalent antibody in multicellular tumour spheroids.

BACKGROUND: New adjuvant immunological therapies that selectively redirect effector cells towards tumour cells are currently under development. These strategies include the use of bispecific antibodies as promising tools for the elimination of disseminated tumour cells and micrometastases. At present, bispecific molecules have demonstrated their antitumour potential in investigations in vitro using monolayer cell cultures. However, their effectiveness in vivo is less clear and expressive in vitro tumour models are in high demand. MATERIALS AND METHODS: Three-dimensional multicellular tumour spheroids (MTS) are of intermediate complexity between monolayer cell cultures and solid tumours in patients and therefore represent a particularly promising in vitro system. RESULTS: We show, here, the antitumour potential of a bispecific antibody, BiUII, in three-dimensional multicellular tumour spheroids and furthermore demonstrate that BiUII triggers peripheral blood mononuclear cells to invade MTSs and elicit the production of TNFalpha, resulting in the efficient destruction of tumour cells. CONCLUSION: These results demonstrate that bispecific antibodies are capable of activating immune effector cells, resulting in the elimination of three-dimensional structures of tumour cells. The therapeutic potential of these antibodies in the clinical setting merits further investigations.

Antibodies, Bispecific↗

PubMedDate not supplied

Prednisolone reduces TNF-alpha release by PBMCs activated with a trifunctional bispecific antibody but not their anti-tumor activity.

BACKGROUND: New adjuvant immunological therapies, that selectively redirect effector cells towards tumors, are currently under development. These strategies include trifunctional bispecific antibodies (trAb) as promising tools for the elimination of disseminated tumor cells and micrometastases. To date, these chimeric molecules have demonstrated their antitumor potential mainly in vitro. Here, trAb-activated peripheral blood mononuclear cells (PBMCs) displayed considerable antitumor activity, accompanied by the release of cytokines, which contributed to the antitumor activity but, on the other hand, may evoke serious limiting side-effects in vivo, demanding therapeutic interventions. MATERIALS AND METHODS: The antitumor activity and cytokine release by trAb-activated PBMCs were studied in co-cultures with multicellular tumor spheroids (MTS), which represent a three-dimensional in vitro model for solid tumors, especially non-vascularized micrometastases. The glucocorticoid prednisolone was tested for its influence on the release of TNF-alpha and the activity of PBMCs. RESULTS: It was shown that PBMCs, which were stimulated with a trifunctional bispecific antibody, BiUII, displayed an excellent antitumor activity, resulting in complete disintegration of the MTS. Also, it was demonstrated that prednisolone significantly reduced the release of TNF-alpha, without impairing the antitumor activity of BiUII-activated PBMCs. In contrast, unspecific killing was reduced, as demonstrated with an identical trAb (Bi48), which recognizes an antigen absent from the target cells. CONCLUSION: The in vivo application of bispecific antibodies for adjuvant tumor therapies may be limited by the manifest activation of immune effectors, accompanied by overwhelming cytokine release. Glucocorticoids, like prednisolone, may effectively reduce cytokine release without impairing the antitumor activity of trAb-activated immune cells.

Animals↗

PubMedDate not supplied

Limited suitability of EpCAM for molecular staging of tumor borders in head and neck cancer.

BACKGROUND: The epithelial cell adhesion molecule (EpCAM) is expressed in most normal epithelia, but is absent from squamous stratified epithelia. However, a de novo expression can be observed in squamous epithelia during carcinogenesis. MATERIALS AND METHODS: In order to evaluate EpCAM as a molecular marker to indicate borders of high risk for the development of local recurrences, its expression was examined in the marginal zone of malignancies. Specimens of squamous cell carcinoma of the head and neck (SCCHN), of the histologically tumor-free defined resection margin and of healthy epithelia of 20 patients were examined by RT-PCR in order to identify the expression of EpCAM in these three different areas. Additionally, immunohistochemistry was perfonned on biopsies from 10 patients in order to confirm these findings and to investigate a potential correlation between EpCAM expression and the degree of dysplasia. RESULTS: By RT-PCR, high expression of EpCAM was found in the tumor. An inverse correlation was observed between EpCAM expression and the distance from the tumor, with no expression being detectable in healthy oral mucosa. In 70% of the cases, EpCAM was expressed in the marginal zone, which had been defined as tumor-free by routine histopathological assessment. Additional immunohistology revealed no correlation between EpCAM expression and the grade of dysplasia. CONCLUSION: Our data provide evidence that EpCAM is restricted as a marker for redefining the real tumor margin by RT-PCR. To complement routine histology, immunohistochemical staining with EpCAM is limited due to its expression in hyperplastic tissue without dysplastic changes. Both observations limit the reliable use of EpCAM for the molecular definition of the critical tumor border and resection margins.

Aged↗

PubMedDate not supplied

Evaluation of CK8-specific autoantibodies in carcinomas of distinct localisations.

Serological screening techniques, such as SEREX and AMIDA, use tumour-specific antibodies in the blood of patients to identify tumour-associated antigens. Using AMIDA, we have recently reported the identification of cytokeratin 8 (CK8) as a new tumour antigen and the presence of elevated serum levels of CK8-specific antibodies (CK8-Abs) in patients with head and neck cancer (HNC). The translation of such a biological observation into a reliable biomarker for clinical diagnosis is a major challenge across the border of biological and clinical research. A prerequisite for a valuable biomarker is specificity, ie. its restriction to a defined disease. Whether the serum levels of CK8-Abs are elevated in patients suffering from other diseases besides HNC was evaluated in this study. A panel of 749 serum samples from patients with carcinomas and benign diseases of different localisations, autoimmune diseases and infections, as well as from clinically healthy donors, was tested. In total, 3.2 times more patients with benign disease (16%) and 2.4 times more patients with carcinomas (12%) showed specific CK8 serum reactivity compared to the healthy donors. However, reactivity was not associated with the kind of disease, i.e. malignant or benign disease, but rather with the disease localisation. Thus CK8-Abs on their own do not allow for the detection of carcinoma of a defined localisation or for the differentiation between carcinoma and benign diseases.

Adult↗