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R Zeidler

Publications and source records attributed to R Zeidler.

At least 37 records · Page 2Linked to original sources
PubMed1997

In vitro studies of liposome-mediated gene transfer into head and neck cancer cell lines.

The 5-year survival rate of patients with squamous cell carcinoma of the head and neck (HNSCC) has remained poor despite innovative surgery and new radiation and chemotherapeutic strategies. In such patients, gene therapy relying on the modification of tumor cells by gene transfer may have great potential as a new treatment modality in the therapy of HNSCC. In the present study we developed an in vitro model to show the efficacy and technical feasibility of cationic liposome-mediated gene transfer into HNSCC. Five adherent squamous cell carcinoma cell lines were transfected with SV40- or CMV-promoter-driven CAT (chloramphenicol-acetyl-transferase)-expression plasmids using DOTAP as the liposome carrier. The level of CAT expression was shown to correlate directly with the amount of transfected DNA and could be measured by a CAT-enzyme-linked immunosorbent assay. The results of gene transfer by liposome-DNA complexes obtained for all cell lines showed a dose-dependent efficacy correlating to the amount of DOTAP employed. The data demonstrate the successful in vitro transfection of epithelial cell lines with DNA, suggesting its usefulness as a new tool for head and neck cancer therapy in vivo.

Carcinoma↗
PubMed1997

[Receptor mediated gene transfer in squamous epithelial carcinoma of the head-neck area].

BACKGROUND: The "Adeno-Virus-Enhanced-Receptor-Mediated" (AVET)-System uses the receptor-mediated endocytosis route to introduce DNA into mammalian cells. By the help of a replication defective adenovirus with endosomolytic activity the coentry of receptor-bound DNA particles is faciliated. The transport to the nucleus followed by gene expression is multifold enhanced. METHODS: Two different permanent cell lines from head and neck squamous cell cancer were transfected with the reportergene for luciferase with either AVET or different liposoms in vitro. RESULTS: In comparison it becomes obvious, that AVET shows a multifold higher transfection rate in vitro than the liposomal formulations applied. CONCLUSIONS: AVET could be used for efficient transfection of epithelial cells, that are fairly reluctant to DNA transfer. Further trials in the mouse model must prove a possible application in vivo.

Adenoviruses, Human↗
PubMed1997

IFN-gamma-mediated coordinated transcriptional regulation of the human TAP-1 and LMP-2 genes in human renal cell carcinoma.

Some human tumor cells exhibit deficient expression of the peptide transporters TAP1 and TAP2 and of the proteasome subunits low molecular weight protein (LMP)-2 and LMP-7, which could be partially restored by cytokine treatment. Here, we show that IFN-gamma stimulation of human renal cell carcinoma lines increased the MHC class I, transporter associated with antigen processing (TAP), and LMP transcript and protein levels, but TAP and LMP expression are more rapidly induced by IFN-gamma than MHC class I molecules. No correlation between the level of induction of the MHC class I antigen presentation genes and IFN sensitivity/resistance was detected. The IFN-gamma-mediated increase of MHC class I, TAP-1, and LMP-2 expression was independent of de novo protein synthesis. Analysis of the dual TAP-1/LMP-2 promoter activity revealed that TAP-1 and LMP-2 expression are controlled by IFN-gamma at the transcriptional level. Site-specific mutations in the IFN-gamma-responsive element of the TAP-1/LMP-2 promoter blocked induction by IFN-gamma. Thus, the IFN-gamma-mediated coordinated transcriptional up-regulation of TAP-1 and LMP-2 expression occurs through the use of a common regulatory element, which might result in enhanced recognition of renal cell carcinoma cells by the immune system.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Epstein-Barr virus latent membrane protein (LMP1) is not sufficient to maintain proliferation of B cells but both it and activated CD40 can prolong their survival.

Epstein-Barr virus (EBV) infects human primary B lymphocytes and induces and maintains proliferation of these cells efficiently in vitro. Mutants of Epstein-Barr virus which express EBV nuclear antigen 2 (EBNA2) in a conditional fashion allow dissection of individual contributions of viral genes to B cell immortalization. EBNA2 is a transcriptional activator of cellular and viral genes, including the viral latent membrane protein 1 (LMP1), which is essential for B cell immortalization and has oncogenic effects in non-lymphoid cells. To analyze the role of this gene in B cell immortalization, LMP1 was constitutively expressed in B cells infected with EBV carrying a conditional EBNA2 allele. In the absence of functional EBNA2, LMP1 was incapable of sustaining B cell proliferation in two independent assays but induced a phenotype consistent with prolonged cell viability. Activation of CD40 displayed a comparable phenotype. These data indicate that both CD40 activation and LMP1 expression may use a common pathway for B cell activation. Proliferation of human B cells, however, requires one or more additional signals triggered by EBNA2.

B-Lymphocytes↗
PubMed1996

Rapid proliferation of B cells from adenoids in response to Epstein-Barr virus infection.

EBV is a human tumor virus that is associated with different types of tumors. A unique feature of EBV is its capability to infect and immortalize human B cells both in vivo and in vitro. In cell culture, this progress is termed immortalization and infected B cells grow out to permanent, so-called lymphoblastoid cell lines. During our experiments, we observed that B lymphocytes derived from adenoids are infected efficiently by EBV and proliferate much more rapidly than any other known type of B cell. High concentrations of adhesion molecules and of CD21, the EBV receptor, present on these cells may account for this phenomenon. Adenoid B cells may therefore represent a particular subpopulation of preactivated B lymphocytes that can greatly simplify and enhance the production of lymphoblastoid cell lines for, e.g., antigen-presenting cells for gene therapeutic approaches and similar applications.

Adenoids↗
PubMed1995

Immortalization of human primary B lymphocytes in vitro with DNA.

Epstein-Barr virus (EBV) is a human DNA tumor virus that efficiently immortalizes human primary B lymphocytes in vitro. Although viral genes that are expressed in latently infected B lymphocytes have been shown to function in cellular growth control, their detailed genetic analysis has been cumbersome for two reasons. The viral genome is too large to permit genetic engineering and human primary B lymphocytes, the only targets for infection by EBV in vitro, are both intractable in culture and recalcitrant to DNA transfection. To overcome these obstacles, we have assembled all the essential genes of EBV on a single recombinant vector molecule in Escherichia coli. We show here that this mini-EBV plasmid can yield immortalized B cells upon transfer of its naked DNA into human primary B lymphocytes. Established cell lines carry recombinant vector DNA and cannot support virus production. Because this DNA can be easily manipulated in E. coli, mutant mini-EBVs as well as foreign genes can now be introduced and studied successfully in recipient B lymphocytes from any human donors. These mini-EBVs therefore are potentially useful for human gene therapy.

B-Lymphocytes↗
PubMed1995

Akv murine leukemia virus enhances lymphomagenesis in myc-kappa transgenic and in wild-type mice.

The contribution of endogenous retroviruses to the multistep process of lymphomagenesis was investigated in wild-type mice and in two different myc-kappa transgenic mouse lines by infection with Akv. This retrovirus is derived from the endogenous ecotropic provirus of the AKR mouse and was previously considered to be nonlymphomagenic. The mice of the two myc-k transgenic lines are predisposed to B-cell lymphomagenesis and were therefore considered to be more susceptible to Akv. For comparison, the same mouse strains were also infected with the exogenous Moloney murine leukemia virus (MoMuLV). Both MoMuLV and Akv increased the tumor incidence and shortened the tumor latency period in wild-type mice and in the transgenic mouse lines. The differences in pathogenicity, number of provirus integrations, and level of virus expression between MoMuLV and Akv indicate different mechanisms of lymphomagenesis: while MoMuLV induced tumors apparently by insertional mutagenesis involving common integration sites similar to previous reports, the enhancement of lymphomagenesis by Akv seems to be directed by other mechanisms.

AKR murine leukemia virus↗
PubMed1995

Integration vectors for antibody chimerization by homologous recombination in hybridoma cells.

Gene targeting in hybridoma cells provides a tool for generating chimeric antibodies with great ease and at high yield. We present an evaluation of integration vectors for the chimerization of the immunoglobulin heavy chain locus which are universally applicable to hybridomas of different isotypes and mouse strains. There are three problems arising with vector integration: (i) the frequent persistence of the parental isotype; (ii) an isotype-dependent aberrant replacement-like recombination giving rise to antibodies devoid of the CH1 domain; and (iii) secondary recombinations leading to excision of the integrated sequence. To overcome these problems, we have systematically evaluated the consequences of extending the vector flank. Although the homology length clearly determines the recombination frequency, this effect is counteracted by the secondary recombination, which also correlates to the homology length. In contrast, the truncating recombination events are not dependent on the homology length and never lead to re-excision of the construct. To take advantage of the increased genetic stability obtained with short flanks, we constructed an enrichment vector which yields high recombination efficiencies despite using a short flanking sequence. In addition, irradiation of the cells enhanced homologous recombination. The problem of the co-production of two isotypes was overcome by a two-step targeting reaction.

Animals↗
PubMed1995

Immortalization of human B lymphocytes by a plasmid containing 71 kilobase pairs of Epstein-Barr virus DNA.

We have assembled derivatives of Epstein-Barr Virus (EBV) that include 71 kbp of noncontiguous DNA sequences cloned into a prokaryotic F-factor plasmid. These mini-EBVs, when introduced into an EBV-containing lymphoblastoid cell, can be packaged by the endogenous helper virus. One such mini-EBV was found to have a single C residue deleted from its EBNA3a open reading frame. When packaged, this mini-EBV initiates proliferation of infected primary human B lymphocytes only in conjunction with a complementing helper virus. Proliferation of the infected cells, however, was maintained either alone by the mini-EBV containing the mutated EBNA3a open reading frame or alone by its derivative in which the EBNA3a open reading frame had been healed of its lesion by recombination with the helper virus. The mini-EBV with a wild-type EBNA3a open reading frame when packaged alone can both initiate and maintain proliferation upon infection of primary human B lymphocytes. These findings identify 41% of EBV DNA which is sufficient to immortalize primary human B lymphocytes and provide an assay to distinguish virus contributions to initiation or maintenance of cell proliferation or both. They also identify EBNA3a as a transforming gene, which contributes primarily to the initiation of cell proliferation.

Amino Acid Sequence↗
PubMed1995

Proliferation-associated nuclear antigen Ki-S1 is identical with topoisomerase II alpha. Delineation of a carboxy-terminal epitope with peptide antibodies.

Proliferation-linked expression of the nuclear Ki-S1 antigen is a significant prognostic indicator in mammary carcinomas. Here, we show staining of a protein of 170 kd by Ki-S1 antibody in immunoblots of Saccharomyces cerevisiae expressing human topoisomerase II alpha but not in the parental strain. In HL-60 cells containing both isoforms of human topoisomerase II, Ki-S1 antibody binds selectively to the 170-kd isoenzyme in a similar fashion as peptide-antibodies directed against amino acid residues 1 to 15 or 1512 to 1530 of human topoisomerase II alpha. Conversely, antibodies directed against carboxyl-terminal sequences of human topoisomerase II beta selectively stain a 180-kd protein. The immunoreactive pattern of V8 endoproteinase restriction digests of human topoisomerase II alpha was identical for Ki-S1-antibody and peptide-antibodies directed against residues 1512 to 1530 but different for peptide-antibodies directed against residues 1 to 15. The Rf values of the smallest fragment commonly recognized by Ki-S1 antibody and the carboxy terminus-specific peptide-antibody place the Ki-S1 epitope within the last 495 carboxyl-terminal amino acid residues of topoisomerase II alpha.

Animals↗
PubMed1995

Unaltered immunoglobulin expression in hybridoma cells modified by targeting of the heavy chain locus with an integration vector.

Chimeric antibodies against the murine T-cell antigen Thy-1.2 were generated in amounts sufficient for in vivo studies by substituting the constant gene segments via homologous recombination in the hybridoma cell. We show that an integration vector targets the heavy chain locus at high frequency even in a non-isogenic situation. Using this vector type, for the first time expression rates were obtained that were identical to the parental hybridoma. The use of the gpt selection marker seems to be crucial for efficient expression, and may overcome a recently claimed drawback of vector integration. A chimeric antibody produced by gene targeting was characterized in vitro and in vivo.

Animals↗
PubMed1994

Breakpoints of Burkitt's lymphoma t(8;22) translocations map within a distance of 300 kb downstream of MYC.

The variant translocation t(8;22) in Burkitt's lymphoma (BL) cells joins band q24 of chromosome 8 distal to the proto-oncogene MYC to the immunoglobulin lambda locus. The distribution of breakpoints on chromosome 8 of 11 cell lines with t(8;22) has been investigated by in situ fluorescence hybridization and pulsed-field gel electrophoresis. We show that these chromosomal breakpoints generally fall within a region of about 300 kb 3' of MYC and that at least 8 out of 11 affect the previously characterized transcriptional unit PVT1. Comparable results were obtained in earlier experiments analyzing the variant t(2;8). Recently, in a series of BL cells carrying t(8;14), breakpoints upstream of MYC have been described at a similar distance. Therefore, our results suggest that deregulation of MYC by the immunoglobulin loci can occur at a distance of up to about 350 kbp of MYC.

Burkitt Lymphoma↗
PubMed1994

[Proliferative activity and defective replication in breast cancer].

The highly proliferating phenotype of mammary carcinoma is known to be associated with a particularly aggressive clinical course. We have been interested in the underlying molecular causes that give rise to the increased proliferative activity Proliferative activity was determined immunohistochemically by the detection of topoisomerase II-alpha (Ki-S1). The subgroup of highly proliferating tumors with a Ki-S1 index exceeding 30% was characterized by a high frequency of c-myc amplification and aberrant p53 expression, whereas tumors, with a low mitotic activity rarely exhibited gene amplification or an altered p53 expression. We conclude, that the highly proliferating phenotype is not capable of regular replication and tends to develop gene amplifications. One of the causes might be a defective cell cycle control by p53.

Breast Neoplasms↗
PubMed1993

Characterization of two novel single-stranded DNA-specific autonomously replicating sequence-binding proteins from Saccharomyces cerevisiae, one of which is adenylosuccinate synthetase.

We report here the identification and characterization of two novel proteis from Saccharomyces cerevisiae that bind to the T-rich strand of the core consensus autonomously replicating sequence (ARS) in a highly specific manner. The two proteins, 40 and 45 kDa in size, can be distinguished by multiple criteria from each other and from the 65-kDa ssArS-T-binding protein identified recently in our laboratory (Schmidt, A. M. A., Herterich, S. U., and Krauss, G. (1991) EMBO J. 10, 981-985). The specificity of binding is inferred from gel shift and nuclease-footprinting experiments using single-stranded probes containing the core consensus ARS. With a 321-nucleotide single-stranded ARS1 fragment, specific protection of the A and B1 domain against DNase I digestion is observed. Partial amino acid sequencing and enzymatic assays identify the 45-kDa protein as adenylosuccinate synthetase, an enzyme necessary for the de novo synthesis of adenylate.

Adenylosuccinate Synthase↗
PubMed1987

[Clinical relevance of immunoglobulin subclasses in children with chronic pulmonary diseases].

In 52 children (age 4-19.5 years; 17 female, 35 male) with chronic chest symptoms IgG-Subclass levels were measured by radial immunodiffusion techniques. Thirteen children had proven bronchiectasis, 22 chronic bronchitis and 17 steroid dependent asthma bronchiale. Cystic fibrosis, alpha-1-antitrypsin deficiency, tuberculosis and chronic foreign bodies were excluded in each patient; all of them showed normal levels of total IgG. Four children (3 with bronchiectasis, one with steroid dependent asthma bronchiale) had a complete lack if IgG4. In two children with chronic bronchitis one showed fluctuating levels of IgG2 and IgG4 and another deficiency of IgG3. All patients with an isolated IgG4 deficiency were treated with immunoglobulins. Of 3 patients with bronchiectasis one improved, two remained unchanged as shown by positive sputum cultures and of chronic chest symptoms. One patient with steroid dependent asthma bronchiale markedly improved during immunoglobulin therapy. It is concluded, that early screening of IgG subclass deficiency is indicated in all children with chronic chest symptoms.

Adolescent↗
PubMed1984

Erythrocyte phosphates and hemoglobin function in monotremes and some marsupials.

Hematologic values, red blood cell (RBC) organic phosphate composition, hemoglobin function, and hemoglobin composition have been determined on blood from the monotremes, the duckbill platypus and the echidna, and three species of marsupials, the Tasmanian devil, the wallaby, and the brush-tail possum. Blood from the platypus had a RBC count of 8.63 X 10(6)/mm3, a mean corpuscular volume of 49.1 millemicron3, and a white blood cell count of 26.0 X 10(3)/mm3. The RBCs from the monotremes and the three marsupials exhibited hemoglobin polymorphism, each with three hemoglobin components. Addition of ATP, 2,3-bisphosphoglycerate (2,3-P2-glycerate), or inositol pentakisphosphate (inositol-P5) to phosphate-free hemoglobin from each species decreased hemoglobin oxygen affinity; the order of effect of these compounds was ATP less than 2,3-P2-glycerate less than inositol-P5. The RBCs of all species had concentrations of 2,3-P2-glycerate ranging from 6.02 mumol/ml RBCs in the wallaby to 10.39 mumol/ml RBCs in the possum. The RBCs from the three species of marsupials had concentrations of ATP ranging from 0.24 mumol/ml RBCs in the possum to 0.80 mumol/ml RBCs in the Tasmanian devil. The level of ATP in RBCs of the platypus and echidna were 0.06 and 0.03 mumol/ml RBCs, respectively.

Adenosine Triphosphate↗