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Hubert Pehamberger

Publications and source records attributed to Hubert Pehamberger.

At least 37 records · Page 2Linked to original sources
PubMed2005

Epitope-specific antibody response to Mel-CAM induced by mimotope immunization.

Peptide mimotopes of tumor antigen epitopes have been proposed as components of tumor vaccines. In this study, we determined the immunogenicity of melcam mim1 and melcam mim2, peptide mimics of an epitope of the melanoma cell-adhesion molecule (Mel-CAM). BALB/c mice were vaccinated either with mimotopes or mimotopes coupled to tetanus toxoid (TT). The antibody responses of mice to melcam mim1, melcam mim2, and recombinant Mel-CAM were analyzed by an ELISA and immunoblot analyses. TT-coupled mimotopes led to high titers of IgG mainly of the IgG2a subclass to melcam mim1 and melcam mim2. Immunization with each of the mimotope formulations induced antibodies that cross-reacted with recombinant Mel-CAM. Uncoupled mimotopes induced lymphocyte proliferation and cytokine production in spleen cell cultures indicating that both peptide mimotopes also contained T cell epitopes. TT-coupled mimotopes induced T helper (Th)1 (interleukin (IL)-2, interferon-gamma) and Th2 (IL-4, IL-5) cytokines, whereas uncoupled mimotopes induced a Th1-biased T cell response. Our results suggest that mimotopes potentially represent a novel vaccine approach to induce a tumor antigen-specific humoral and cellular response.

Amino Acid Sequence↗
PubMed2005

The non-receptor-associated tyrosine kinase Syk is a regulator of metastatic behavior in human melanoma cells.

Melanoma is one of the most aggressive neoplastic transformations and characterized by a high metastatic potential. The current study was performed to assess the impact of "spleen tyrosine kinase" (Syk), a non-receptor-associated tyrosine kinase, on growth and metastatic behavior of melanoma cells in vitro and in a severe combined immunodeficient (SCID)-mouse/human-melanoma xenotransplantation model in vivo. Syk was expressed in melanocytes but was found to be downregulated in melanoma cells. Vector-driven expression of Syk in two different melanoma cell lines did not influence growth speed, but significantly reduced the invasive growth potential of both cell lines in a Matrigel assay in vitro. In a SCID-mouse/human melanoma xenotransplantation model, Syk expressing Mel-Juso cells exhibited delayed and reduced tumor growth. After intravenous as well as subcutaneous injection of tumor cells, Syk-transfected cells formed significantly fewer metastatic tumor lesions than control cells. The presented data define Syk as a novel regulator of metastatic behavior of melanoma cells.

Animals↗
PubMed2005

Clusterin regulates drug-resistance in melanoma cells.

Clusterin has recently been shown to act as an antiapoptotic protein that confers drug-resistance in models of epithelial tumors. The aim of our work was to provide an insight into a possible role of clusterin in the regulation of drug-resistance in melanoma. In tissue samples, clusterin expression was low in nevi, but high in primary melanoma and melanoma metastases. Clusterin was also strongly expressed in melanoma cell lines, but was barely detectable in cultured melanocytes. To elucidate a possible role of clusterin in drug-resistance of melanoma, clusterin expression was regulated by either plasmid-driven overexpression or by antisense-mediated downregulation. Clusterin overexpression was associated with an increase in drug-resistance, i.e., with an increased survival of melanoma cells in the presence of cytotoxic drugs. In contrast, downregulation of clusterin by 2'-O-(2-methoxy)ethyl (2'MOE)-modified antisense oligonucleotides (AS-ODN) directed against clusterin mRNA significantly reduced drug-resistance, i.e., decreased survival of melanoma cells in the presence of cytotoxic drugs. To evaluate the effects of clusterin-antisense treatment in vivo, we applied an SCID-mouse/human-melanoma xenotransplantation model. Pre-treatment of mice with the 2'MOE-modified clusterin AS-ODN was associated with a significantly improved tumor response to dacarbazine as compared with animals pretreated with a scrambled control oligonucleotide. Taken together, we show that clusterin is strongly expressed in melanoma. Downregulation of clusterin reduces drug-resistance, i.e., reduces melanoma cell survival in response to cytotoxic drugs in vitro and in vivo. Thus, reducing clusterin expression may provide a novel tool to overcome drug-resistance in melanoma.

Animals↗
PubMed2005

Thalidomide enhances the anti-tumor activity of standard chemotherapy in a human melanoma xenotransplatation model.

It has been demonstrated that thalidomide's anti-angiogenic properties result in clear anti-tumor activity in a number of human malignancies. We studied thalidomide in a human melanoma severe combined immunodeficiency mouse xenotransplantation model. Thalidomide as a single agent showed a significant tumor reduction of 46% compared with the control group. Thalidomide combined with dacarbazine treatment markedly enhanced the anti-tumor effect of chemotherapy and showed a significant tumor reduction relative to the dacarbazine-only group (61%) and even more tumor reduction (74%) compared with the control group. We also measured clearly reduced levels of tumor necrosis factor-alpha in the thalidomide-treated group. A significantly lower microvessel density was encountered in the thalidomide treatment groups (thalidomide alone or combined with DTIC), underscoring the anti-angiogenic effect of thalidomide as a single agent as well as in combination with chemotherapy in this model. In line with these results, we observed a nearly 3-fold increase of apoptosis for the combination of thalidomide and DTIC compared with the rate of apoptotic cells in DTIC-only-treated melanoma xenotransplants. These data underline the rationale for combining dacarbazine--a cytotoxic agent--and thalidomide--an anti-angiogenic cytostatic agent--as a promising strategy for the treatment of melanoma.

Angiogenesis Inhibitors↗
PubMed2005

Seven novel and stable translocations associated with oncogenic gene expression in malignant melanoma.

Cytogenetics has not only precipitated the discovery of several oncogenes, but has also led to the molecular classification of numerous malignancies. The correct identification of aberrations in many tumors has, however, been hindered by extensive tumor complexity and the limitations of molecular cytogenetic techniques. In this study, we have investigated five malignant melanoma (MM) cell lines from at least three different passages using high-resolution R-banding and the recently developed methods of comparative genomic hybridization and multicolor or multiplex fluorescence in situ hybridization. We subsequently detected nine consistent translocations, seven of which were novel: dic(1;11)(p10;q14), der(9)t(3;9)(p12;p11), der(4)t(9;4;7)(q33:p15-q23:q21), der(14)t(5;14)(q12;q32), der(9)t(9;22)(p21;q11), der(19)t(19;20)(p13.3;p11), der(10)t(2;12;7;10)(q31:p12-->pter:q11.2-->q31:q21), der(19)t(10;19)(q23;q13), and der(20)t(Y;20)(q11.23;q13.3). Furthermore, using the human HG-U133A GeneChip, positive expression levels of oncogenes or tumor-related genes located at the regions of chromosomal breakpoints were identified, including AKT1, BMI1, CDK6, CTNNB1, E2F1, GPNMB, GPRK7, KBRAS2, LDB2, LIMK1, MAPK1, MEL, MP1, MUC18, NRCAM, PBX3, RAB22A, RAB38, SNK, and STK4, indicating an association between chromosomal breakpoints and altered gene expression. Moreover, we also show that growth of all five cell lines can be significantly reduced by downregulating CDK6 gene expression with small interfering RNA (siRNA). Because the majority of these breakpoints have been reported previously in MM, our results support the idea of common mechanisms in this disease.

Cell Line↗
PubMed2004

High-molecular-weight melanoma-associated antigen mimotope immunizations induce antibodies recognizing melanoma cells.

Size and posttranslational modifications are obstacles in the recombinant expression of high-molecular-weight melanoma-associated antigen (HMW-MAA). Creating a tumor antigen mimic via the phage display technology may be a means to overcome this problem for vaccine design. In this study, we aimed to generate an immunogenic epitope mimic of HMW-MAA. Therefore we screened a linear 9mer phage display peptide library, using the anti-HMW-MAA monoclonal antibody (mAb) 225.28S. This antibody mediates antibody-dependent cellular cytotoxicity (ADCC) and has already been used for anti-idiotype therapy trials. Fifteen peptides were selected by mAb 225.28S in the biopanning procedure. They share a consensus sequence, but show only partial homology to the amino acid sequence of the HMW-MAA core protein, indicating mimicry with a conformational epitope. One mimotope was chosen to be fused to albumin binding protein (ABP) as an immunogenic carrier. Immunoassays with 225.28S indicated that the mimotope fusion protein was folded correctly. Subsequently, the fusion protein was tested for immunogenicity in BALB/c mice. The induced anti-mimotope antibodies recognized HMW-MAA of 518A2 human melanoma cells, whereas sera of mice immunized with the carrier ABP alone showed no reactivity. These anti-mimotope antibodies were capable of inducing specific lysis of 518A2 melanoma cells in ADCC assays with murine effector cells. In conclusion, the presented data indicate that mimotopes fused to an immunogenic carrier are suitable tools to elicit epitope-specific anti-melanoma immune responses.

Albumins↗
PubMed2004

Generation of Peptide mimics of the epitope recognized by trastuzumab on the oncogenic protein Her-2/neu.

Immunizations with the oncogenic protein Her-2/neu elicit Abs exerting diverse biological effects--depending on epitope specificity, tumor growth may be inhibited or enhanced. Trastuzumab (herceptin) is a growth-inhibitory humanized monoclonal anti-Her-2/neu Ab, currently used for passive immunotherapy in the treatment of breast cancer. However, Ab therapies are expensive and have to be repeatedly administered for long periods of time. In contrast, active immunizations produce ongoing immune responses. Therefore, the study aims to generate peptide mimics of the epitope recognized by trastuzumab for vaccine formulation, ensuring the subsequent induction of tumor growth inhibitory Abs. We used the phage display technique to generate epitope mimics, mimotopes, complementing the screening Ab trastuzumab. Five candidate mimotopes were isolated from a constrained 10 mer library. These peptides were specifically recognized by trastuzumab, and showed distinctive mimicry with Her-2/neu in two experimental setups. Subsequently, immunogenicity of a selected mimotope was examined in BALB/c mice. Immunizations with a synthetic mimotope conjugated to tetanus toxoid resulted in Abs recognizing Her-2/neu in a blotted cell lysate as well as on the SK-BR-3 cell surface. Analogous to trastuzumab, the induced Abs caused internalization of the receptor from the cell surface to endosomal vesicles. These results indicate that the selected mimotopes are suitable for formulation of a breast cancer vaccine because the resulting Abs show similar biological features as trastuzumab.

Animals↗
PubMed2004

Mcl-1 is a novel therapeutic target for human sarcoma: synergistic inhibition of human sarcoma xenotransplants by a combination of mcl-1 antisense oligonucleotides with low-dose cyclophosphamide.

PURPOSE: Little is known about the role that Mcl-1, an antiapoptotic Bcl-2 family member, plays in solid tumor biology and susceptibility to anticancer therapy. We observed that the Mcl-1 protein is widely expressed in human sarcoma cell lines of different histological origin (n = 7). Because the expression of antiapoptotic Bcl-2 family proteins can significantly contribute to the chemoresistance of human malignancies, we used an antisense strategy to address this issue in sarcoma. EXPERIMENTAL DESIGN: SCID mice (n = 6/group) received s.c. injections of SW872 liposarcoma cells. After development of palpable tumors, mice were treated by s.c.-implanted miniosmotic pumps prefilled with saline or antisense or universal control oligonucleotides (20 mg/kg/day for 2 weeks). On days 2, 6, and 10, mice were treated with low-dose cyclophosphamide (35 mg/kg i.p) or saline control. During the experiments, tumor weight was assessed twice weekly by caliper measurements. On day 14, animals were sacrificed. Tumors were weighed and fixed in formalin for immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling analysis. RESULTS: Mcl-1 antisense oligonucleotides specifically reduced Mcl-1 protein expression but produced no reduction in tumor weight compared with saline-treated control animals. Cyclophosphamide monotreatment caused only modest tumor weight reduction compared with saline control. However, use of Mcl-1 antisense oligonucleotides combined with cyclophosphamide clearly enhanced tumor cell apoptosis and significantly reduced tumor weight by more than two-thirds compared with respective control treatments. CONCLUSION: A combination of Mcl-1 antisense oligonucleotides with low-dose cyclophosphamide provides a synergistic antitumor effect and might qualify as a promising strategy to overcome chemoresistance in human sarcoma.

Animals↗
PubMed2004

Interferon resistance promotes oncolysis by influenza virus NS1-deletion mutants.

NS1 protein of influenza virus is a virulence factor that counteracts Type I interferon (IFN)-mediated antiviral response by the host. A recombinant influenza A virus that lacks the NS1 protein only replicates efficiently in systems that contain defective IFN pathways. We demonstrate that the conditional replication properties of NS1-modified influenza A virus mutants can be exploited for the virus-mediated oncolysis of IFN-resistant tumor cells. IFN resistance in analyzed tumor cell lines correlated with a reduced expression of STAT1. Addition of exogenous IFNalpha or supernatant of virus-infected endothelial cells inhibited viral oncolysis in IFN-sensitive but not in IFN-resistant cell lines. The oncolytic potential of NS1-modified influenza A virus mutants could be exploited in vivo in a SCID mouse model of a subcutaneously-implanted human IFN-resistant melanoma. The data indicate that IFN-resistant tumors are a suitable target for oncolysis induced by NS1-modified influenza virus mutants. STAT1 might serve as a marker to identify these IFN-resistant tumors.

Animals↗
PubMed2004

A novel concept for ligand attachment to oligonucleotides via a 2'-succinyl linker.

Conjugation of ligands to antisense oligonucleotides is a promising approach for enhancing their effects. In this report, a new method for synthesizing oligonucleotide conjugates is described. 2'-Amino-2'-deoxy-5'-dimethoxytrityl-uridine was select ively acylated with a succinic acid linker at the 2' position. This compound was incorporated at the 3' end of an oligonucleotide corresponding to the sequence of Oblimersen. The carboxyl group was protected for oligonucleotide synthesis as a benzyl ester, which could be selectively cleaved at the solid phase by a catalytic phase transfer reaction using palladium nanoparticles as catalyst. An oligonucleotide-fluorescein conjugate was prepared by condensation of aminofluorescein. Circular dichroism spectroscopic experiments showed a B-DNA type structure. The melting temperature of the duplex was only slightly lower than that of Oblimersen. Biological activity measured by western blotting resulted in a Bcl-2 target downregulation nearly identical to that of control Oblimersen on human melanoma cells, proving that this method is attractive for the binding of ligands located in the minor groove.

Base Sequence↗
PubMed2004

CpG oligonucleotides elicit antitumor responses in a human melanoma NOD/SCID xenotransplantation model.

For patients with advanced malignant melanoma, the 5 y survival rate with current treatment modalities is low. There is an urgent need for more effective therapeutic concepts. One approach with great potential is to stimulate the body's own immune defense to reject cancer cells using CpG oligonucleotides. Distinct oligonucleotides containing nonmethylated cytidine residues in cytidine-guanosine dinucleotides with particular flanking bases (CpG motifs) are capable of eliciting powerful immune stimulation by mimicking infectious disease. We evaluated the in vivo antitumoral effects of CpG oligonucleotides against human malignant melanoma xenografts in NOD/SCID mice. CpG oligonucleotides administered in single peritumoral subcutaneous injections three times per week resulted in elevated plasma levels of interleukin-12 and significant inhibition of the growth of established tumor xenografts by 60% (p<0.016) compared to the saline control. In addition to this a significant invasion of macrophages into tumor xenografts and increased numbers of Langerhans-cell-derived dendritic cells in draining lymph nodes could be observed. Our findings demonstrate the antitumor activity of oligonucleotides containing immune-stimulatory CpG motifs in a xenotransplantation model with absent B, T cells and a lack of natural killer cell function.

Animals↗
PubMed2004

Loss of novel mda-7 splice variant (mda-7s) expression is associated with metastatic melanoma.

Expression of melanoma differentiation associated gene-7 (mda-7) also known as interleukin 24 (IL-24) decreases during melanoma cell differentiation and induces apoptosis in melanoma cells but not in melanocytes. Here we identify a novel splice variant of the cancer growth suppressor gene mda-7/IL-24 (mda-7s) that is differentially expressed in RNA preparations from normal human melanocytes, transformed melanocytes, nevi, subcutaneous metastasis, lymph node metastasis, and melanoma cell lines. The 450 bp mda-7s mRNA encodes a protein of 63 residues with a molecular weight of 12 kDa. mda-7s lacks exons 3 and 5 of the full-length transcript and contains only 14 amino acids of homology to MDA-7 located within the signal peptide region of the wild-type sequence. Despite minimal homology, MDA-7S coprecipitates full length MDA-7 and reduces secretion of cotransfected MDA-7. mda-7 and mda-7s are coexpressed in all RNA preparations other than subcutaneous and lymph node metastasis where mda-7s expression is lacking. mda-7s expression is therefore linked to a non-metastatic phenotype.

Alternative Splicing↗
PubMed2004

Pegylated and conventional interferon-alpha induce comparable transcriptional responses and inhibition of tumor growth in a human melanoma SCID mouse xenotransplantation model.

Interferon-alpha (IFN-alpha) is widely used for the treatment of viral infections and primary cancers. In the present study, we investigated whether the anti-proliferative activity of IFN-alpha is capable of inhibiting melanoma tumor development in the absence of protective immune responses in a severe combined immunodeficiency (SCID) mouse model. Mice treated with either regular (100 microg/3 times per week) or pegylated (300 microg/once weekly) human IFN-alpha 2a showed a marked reduction in tumor weight after 4 wk of treatment. Tumor weight in pegylated and conventional IFN-alpha-treated animals was reduced by 61% and 67%, respectively, as compared to saline control (both p< or =0.01). A decrease of proliferation and an increase of apoptotic tumor cells were observed in IFN-treated tumors. DNA microarrays were applied to analyze transcriptional responses in tumors after 4 wk of treatment and a subset of about 90 genes was differentially expressed. Twenty-four novel and five known interferon-inducible genes were up- and 65 genes downregulated. A direct comparison of IFN-alpha and pegylated IFN-alpha did not reveal any significant differences in tumor growth inhibition indicating that this novel and more stable class of IFN is functionally equivalent. Despite the structural difference between pegylated and conventional IFN-alpha, both agents caused similar transcriptional responses in human melanoma xenotransplants.

Animals↗
PubMed2004

Comparative histopathology of grey-horse-melanoma and human malignant melanoma.

Equine melanoma shows striking features particularly with regard to clinical development in grey horses: in contrast to malignant melanoma in humans and in solid coloured horses that are characterized by early onset of metastasis, pigment cell tumours display almost benign clinical features in ageing grey horses. Through evolution, grey horses appear to be in a favourable position in regard to the biological behaviour of melanomas. Yet unknown factors inhibiting or retarding early melanoma metastasis may be responsible for this phenomenon. In this study, immunostaining profiles and histopathologic patterns of equine vs. human melanotic tumours were compared. In addition, the expression of melanoma markers currently used in human melanoma detection and characterization were evaluated for their applicability in equine melanoma diagnosis. Immunohistopathologic investigations revealed that benign grey horse melanomas share common features with human blue nevi and with human malignant desmoplastic melanomas, whereas their resemblance to other types of human cutaneous malignant melanomas is less pronounced. Our data equally underline that S-100, proliferating cell nuclear antigen (PCNA), HMB-45, Ki-67, T-311 and CD44 can serve as reliable markers for horse melanomas. Further investigations aiming at identifying factors retarding metastasis in affected grey horses are needed, as they may contribute to the development of novel treatment strategies for human malignant melanoma.

Animals↗
PubMed2004

NF-kappaB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression.

The transcription factor NF-kappaB is activated in a range of human cancers and is thought to promote tumorigenesis, mainly due to its ability to protect transformed cells from apoptosis. To investigate the role of NF-kappaB in epithelial plasticity and metastasis, we utilized a well-characterized in vitro/in vivo model of mammary carcinogenesis that depends on the collaboration of the Ha-Ras oncoprotein and TGF-beta. We show here that the IKK-2/IkappaBalpha/NF-kappaB pathway is required for the induction and maintenance of epithelial-mesenchymal transition (EMT). Inhibition of NF-kappaB signaling prevented EMT in Ras-transformed epithelial cells, while activation of this pathway promoted the transition to a mesenchymal phenotype even in the absence of TGF-beta. Furthermore, inhibition of NF-kappaB activity in mesenchymal cells caused a reversal of EMT, suggesting that NF-kappaB is essential for both the induction and maintenance of EMT. In line with the importance of EMT for invasion, blocking of NF-kappaB activity abrogated the metastatic potential of mammary epithelial cells in a mouse model system. Collectively, these data provide evidence of an essential role for NF-kappaB during distinct steps of breast cancer progression and suggest that the cooperation of Ras- and TGF-beta-dependent signaling pathways in late-stage tumorigenesis depends critically on NF-kappaB activity.

Animals↗
PubMed2003

Inhibition of tumor cell growth by antibodies induced after vaccination with peptides derived from the extracellular domain of Her-2/neu.

The anti Her-2/neu monoclonal antibody Trastuzumab has strong inhibiting effects on tumor growth in vitro and in vivo and is therefore used for immunotherapy in breast cancer patients. Due to necessity of frequent applications, however, cost intensiveness of Trastuzumab treatment and its limited duration of affectivity, an active immunization inducing a perhaps preventive and long-term immunity to Her-2/neu remains a desirable goal. We attempted to induce anti Her-2/neu antibodies by peptide vaccination and to test their efficacy in inhibiting tumor cell growth in vitro. By computer aided analyses, 7 putative B cell epitopes of Her-2/neu were defined and synthesized. These peptide epitopes were coupled to tetanus toxoid and used for immunization in BALB/c mice. Among these peptides, immunizations with 2 single peptides or a combination of 2 peptides induced anti-peptide antibody levels, primarily of the IgG1 isotype. These antibodies were also directed against the native Her-2/neu antigen, as shown in precipitation assays and ELISA with cell lysates of the Her-2/neu overexpressing breast cancer cell line SK-BR-3. Isolated IgG fractions from immune sera incubated with SK-BR-3 cells led to a moderate inhibition of the tumor cell growth in vitro, as well as to complement dependent cell lyses comparable to that achieved by incubation with Trastuzumab. Moreover, peptide immunization in rabbits generated anti-Her 2-neu IgG that, in contrast to mouse sera, were able to mediate a 31-46% lysis of SK-BR-3 cells in ADCC experiments. We conclude from our data that immunization with Her-2/neu peptides successfully induced humoral immune response with anti-tumor activity in an animal model.

Amino Acid Sequence↗
PubMed2003

An endogenous retrovirus derived from human melanoma cells.

We show that human melanoma cells produce retrovirus-like particles that exhibit reverse transcriptase activity, package sequences homologous to human endogenous retrovirus K (HERV-K), and contain mature forms of the Gag and Env proteins. We also demonstrate expression of the pol gene and of Gag, Env, and Rec proteins in human melanomas and metastases but not in melanocytes or normal lymph nodes. The data suggest that expression of retroviral genes and production of retroviral particles is activated during development of melanoma.

Base Sequence↗