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J Gebert

Publications and source records attributed to J Gebert.

15 recordsLinked to original sources
PubMed1999

Dominant negative effect of the APC1309 mutation: a possible explanation for genotype-phenotype correlations in familial adenomatous polyposis.

Inactivation of the adenomatous polyposis coli (APC) gene product initiates colorectal tumorigenesis. Patients with familial APC (FAP) carry germ-line mutations in the APC gene and develop multiple colorectal adenomas and subsequent carcinomas early in life. The severity of the disease correlates with the position of the inherited APC mutation (genotype-phenotype correlation). Together with the fact that both germ-line and sporadic APC mutations cluster in the central region of the APC gene, this points to a dominant negative effect of certain APC mutants. Loss of APC function was recently shown to result in enhanced beta-catenin-/Tcf-mediated transcription in colon epithelial cells. Here, we provide experimental evidence for a dominant negative effect of APC gene products associated with severe polyposis. Wild-type APC activity in beta-catenin-/Tcf-mediated transcription was strongly inhibited by a mutant APC that is truncated at codon 1309. In contrast, mutant APC gene products that are associated with attenuated polyposis (codon 386 or 1465) interfered only weakly with wild-type APC activity. These results suggest a molecular explanation for the genotype-phenotype correlation in FAP patients and support the idea that colorectal tumor growth might be, in part, driven by selection for a mutation in the mutation cluster region.

Adenomatous Polyposis Coli↗
PubMed1999

Molecular screening of potential HNPCC patients using a multiplex microsatellite PCR system.

Microsatellite instability (MSI) is a molecular hallmark of the H ereditary N on- P olyposis C olorectal C ancer (HNPCC) syndrome occurring in about 80-90% of the tumours and also in sporadic tumours of different organs, albeit at lower frequency. Highly unstable colorectal tumours (MSI-H) have different histopathological features and tend to have a better prognosis compared to neoplasms without (MSS) or with low levels of microsatellite instability (MSI-L). Since MSI classification allows the identification of potential HNPCC patients and might represent a valuable diagnostic parameter an increasing demand for high-throughput microsatellite analysis will arise. Therefore, we have adapted five diagnostic microsatellites, m(odified) ACTC, mBAT26, mD5S107, mD5S406 and mD13S153, to allow coamplification. Using this multiplex polymerase chain reaction (PCR) system 29 colorectal tumour tissues with known MSI status could be unambiguously identified as MSI-H (13 cases) or MSI-L/MSS (16 cases). Highly unstable colorectal tumour detection frequency of individual markers reached 77% (mD5S406), 85% (mACTC), 85% (mD5S107), 92% (D13S153) and 100% (mBAT26) showing similar sensitivity but improved specificity as compared with a microsatellite reference panel. In a prospective analysis of 31 colorectal tumours, the multiplex PCR system identified five MSI-H cases. Multiplex MSI PCR is a time saving and cost-effective method not restricted to specific technical equipment and applicable to a variety of microsatellite-based genotyping approaches.

Colorectal Neoplasms↗
PubMed1998

Double suicide gene (cytosine deaminase and herpes simplex virus thymidine kinase) but not single gene transfer allows reliable elimination of tumor cells in vivo.

Suicide genes such as cytosine deaminase (CD) and herpes simplex virus thymidine kinase (TK) encode products that convert nontoxic substances (prodrugs) into toxic metabolites. Suicide gene transfer is currently being used in cancer therapy or can be used as a safety modality. To analyze the reliability of suicide genes as a safety modality for a vaccination study with viable cytokine/B7 gene-modified tumor cells, the individual and combined efficacy of the two suicide genes was compared for in vitro and in vivo cell killing of a murine mammary adenocarcinoma cell line (TS/A). To adapt the system to an in vivo gene delivery situation, bulk cultures cotransfected with the CD and TK gene were used instead of selected clones. In vitro, both CD and TK conferred sensitivity to the respective prodrug but the combined cytotoxic effects of both gene products were always superior. For in vivo analysis BALB/c mice were injected subcutaneously with CD- and TK-modified TS/A cells, treated with prodrugs, and tumor size was evaluated for a period of 100 days. In the in vivo situation the combination of both enzyme/prodrug systems was again most effective. The highest single concentration of 5-FC (500 mg/kg) or GCV (100 mg/kg) was not able to fully protect the animals from developing tumors, whereas a combination of 5-FC (250 mg/kg) and GCV (50 mg/kg) resulted in complete tumor eradication. In nude mice treated in the same way, most CD/TK tumors could not be eliminated. Furthermore, BALB/c mice cured of TS/A-CD/TK tumors developed a systemic tumor immunity against challenge with parental TS/A cells. These findings indicate that reliable tumor elimination by the suicide genes depends on T cells. The cooperative effect of both suicide genes was confirmed in vitro with the human renal cell carcinoma line RCC26. We conclude that TK and CD together, but neither gene alone, act as a safety mechanism for the elimination of tumor cells in a reliable fashion and suggest that a rapid and quantitative antigen release by effective TK- and CD-mediated tumor destruction is necessary for T cell immunity to develop.

Adenocarcinoma↗
PubMed1998

Optimized non-radioactive protein truncation test for mutation analysis of the adenomatous polyposis coli (APC) gene.

Germline mutations in the adenomatous polyposis coli gene cause familial adenomatous polyposis, a colon cancer predisposition syndrome. More than 95% of the identified mutations result in the generation of stop codons or reading frame shifts and encode a truncated gene product, a mutation profile also found in other tumor predisposition genes such as the breast cancer or the hereditary non-polyposis coli. Therefore the protein truncation test is ideally suited for screening of mutations in these genes, starting from simple blood samples. Gene segments of interest are amplified from genomic DNA or mRNA, thereby incorporating a T7 promoter at the 5'-end. After in vitro transcription and translation of the PCR products, the resulting protein is analysed by gel electrophoresis. Truncated translation products indicate the presence of a stop mutation. We have developed a non-radioactive protein truncation test that uses a biotinylated Lys-t-RNA to label the translation products and allows a chemiluminescent detection instead of the standard radioactive method. This generic protein truncation test kit was then used to develop a parameter-specific protein truncation test for adenomatous polyposis coli. The adenomatous polyposis coli gene was divided in 5 overlapping segments, and primers were optimized to produce distinct bands with very low background in the protein truncation test. The assay was tested on 20 familial adenomatous polyposis patient samples, where 18 mutations were found, demonstrating the efficiency of this method.

Adenomatous Polyposis Coli↗
PubMed1998

[Modification of surgical strategy in HNPCC by molecular and clinical aspects].

Because of the positive correlation between Amsterdam criteria and positive MSI analysis, a subtotal colectomy with ileorectal anastomosis seems to be indicated in patients with positive Amsterdam criteria to eliminate the high risk of metachronous colon cancer. In patients with an identified mutation in one of the known mismatch repair genes, a subtotal colectomy is indicated as well. In patients with positive Bethesda criteria, apart from the Amsterdam criteria, a subtotal colectomy seems only to be indicated if a positive MSI analysis is available.

Adult↗
PubMed1998

[Mutation localization as a guide for surgical approach in familial adenomatous polyposis?].

Restorative proctocolectomy and ileal pouch-anal anastomosis (IPAA) is considered the operative therapy of choice for the prophylactic treatment of FAP. Recently, Vasen and coworkers [5] after correlating the incidence of metachronous rectal cancer with the site of the causative APC mutation suggested subtotal colectomy and IRA to be the primary treatment in patients with mutations proximal to codon 1250, whereas IPAA should be performed in those with mutations beyond this codon. Mutation analysis in our patients after IRA, however, shows the majority of APC mutations to be located proximal to codon 1250 even in those patients with severe rectal polyposis and metachronous rectal cancer, thus not supporting the therapeutic recommendations of Vasen and coworkers.

Adenomatous Polyposis Coli↗
PubMed1997

Microsatellite instability analysis: a multicenter study for reliability and quality control.

The molecular biology section of the Hereditary Non-Polyposis Colorectal Cancer study group-Germany, instituted a multicenter study to test the reliability and quality of microsatellite instability (MSI) analysis. Eight laboratories compared MSI analyses performed on 10 matched pairs of normal and tumor DNA from patients with colorectal carcinomas. A variety of techniques were applied to the detection of microsatellite changes: (a) silver and ethidium bromide staining of polyacrylamide gels; (b) radioactive labeling; and (c) automated fluorescence detection. The identification of highly unstable tumors and tumors without MSI was achieved in high concordance. However, the interpretation of the band patterns resulted in divergent classifications at several microsatellite marker loci for a large fraction of this tumor/normal panel. The data on more than 30 primers per case suggest that the enlargement of the microsatellite panel to more than 10 loci does not influence the results. In this study, cases with MSI in less than 10% of loci were classified as microsatellite stable, whereas MSI was diagnosed in cases with more than 40% of all markers unstable. We propose that a panel of five microsatellite loci consisting of repeats with different lengths should be analyzed in an initial analysis. When less than two marker loci display shifts in the microsatellite bands from tumor DNA, the panel should be enlarged to include an additional set of five marker loci. The number of marker loci analyzed as well as the number of unstable marker loci found should always be identified. These criteria should result in reports of MSI that are more comparable between studies.

Chromosome Deletion↗
PubMed1996

Immune reactions induced by interleukin-2 transfected colorectal cancer cells in vitro: predominant induction of lymphokine-activated killer cells.

One aim of the genetic modification of tumor cells is the generation of immunogenic variants that can be used for the induction of immune responses against tumors. We engineered the human colorectal carcinoma cell line SW480 by means of plasmid transfection to secrete interleukin (IL)-2. Transfection of SW480 cells resulted in stable IL-2 secretion at 5-30 ng/ml per 10(5) cells in 24 h and, unexpectedly, in CD54 expression on the cell surface. SW480 variants expressing IL-2 and CD54 were tested for their capacity to induce T lymphocyte activation in vitro in comparison to untransfected and CD54 transfected cells. The cytolytic effector function of a class I MHC restricted CD8+, peptide antigen specific T cell clone was augmented following expression of CD54. IL-2 secreting SW480 variants did not further increase antigen-dependent cytolysis. Primary activation of resting T lymphocytes was assessed following allogeneic stimulation. When compared with unmodified SW480 cells, CD54 expressing variants did not initiate T cell proliferation. In contrast, IL-2 secreting SW480 cells strongly promoted primary T cell proliferation. Similarly, exogenous IL-2 and SW480 cells induced T cell proliferation which was not only due to IL-2 but was dependent on tumor cells. However, following the initial wave of cell growth in response to IL-2 secreting SW480 cells T lymphocytes could not be restimulated with SW480 or IL-2 secreting variants and could not be further expanded. T cells initially activated by IL-2 secreting SW480 cells exhibited cytolytic activity towards SW480 cells. This reactivity, however, was transient and completely blocked by K562 cells, suggesting MHC-unrestricted, nonspecific cytotoxicity. We conclude that endogenous IL-2 secretion by the colorectal carcinoma cell line SW480 does not result in the activation of MHC restricted specific T lymphocytes but predominantly induces lymphokine-activated killer cells. Considering that tumor cell vaccines are aimed at inducing tumor-specific immune responses, our in vitro observation would rather argue against the in vivo application of such a tumor cell modification in colorectal cancer.

Amino Acid Sequence↗
PubMed1996

[Effect of molecular diagnostic procedures on surgical therapy of malignant diseases].

In the past molecular biological techniques have provided the basis for principally new aspects in the diagnosis of neoplastic diseases. The genetic predisposition to hereditary cancer syndromes can be detected by germ line DNA sequence analysis and offers the opportunity for prophylactic surgery. Some preneoplastic lesions can be detected through the identification of specific molecular alterations in nucleic acid preparations derived from various clinical samples. Residual or disseminated tumor cells can be detected in resection margins, lymph nodes, bone marrow, or peripheral blood with great sensitivity. Most likely, these techniques will strongly influence cancer screening programs and provide a rational basis for adjuvant systemic or regional therapy modalities. However, the clinical value of these techniques has not yet been proven in controlled trials. The problems of reproducibility and quality assurance have to be addressed and solved. This review summarizes the basic principles of some of these new molecular diagnostic techniques to permit a critical assessment of their clinical implications.

Cell Transformation, Neoplastic↗
PubMed1996

Adenovirus-assisted lipofection: efficient in vitro gene transfer of luciferase and cytosine deaminase to human smooth muscle cells.

Smooth muscle cells (SMC) are a central cell type involved in multiple processes of coronary artery diseases including restenosis and therefore are major target cells for different aspects of gene transfer. Previous attempts to transfect primary arterial cells using different techniques like liposomes, CaPO4 and electroporation resulted in only low transfection efficiency. The development of recombinant adenoviruses dramatically improved the delivery of foreign genes into different cell types including SMC. However, cloning and identification of recombinants remain difficult and time-consuming techniques. The present study demonstrates that a complex consisting of reporter plasmid encoding firefly luciferase (pLUC), polycationic liposomes and replication-deficient adenovirus was able to yield very high in vitro transfection of primary human smooth muscle cells under optimized conditions. The technique of adenovirus-assisted lipofection (AAL) increases transfer and expression of plasmid DNA in human smooth muscle cells in vitro up to 1000-fold compared to lipofection. To verify the applicability of AAL for gene transfer into human smooth muscle cells we studied a gene therapy approach to suppress proliferation of SMC in vitro, using the prokaryotic cytosine deaminase gene (CD) which enables transfected mammalian cells to deaminate 5-fluorocytosine (5-FC) to the highly toxic 5-fluorouracil (5-FU). The effect of a transient CD expression on RNA synthesis was investigated by means of a cotransfection with a RSV-CD expression plasmid and the luciferase reporter plasmid. Western blot analysis demonstrated high expression of CD protein in transfected SMC. Cotransfected SMC demonstrated two-fold less luciferase activity in the presence of 5-FC (5 mmol/l) after 48 h compared to cells transfected with a non-CD coding plasmid. The data demonstrate that a transient expression of CD could be sufficient to reduce the capacity of protein synthesis in human SMC. This simple and effective in vitro transfection method may also be applicable to in vivo delivery of target genes to the vascular wall to inhibit SMC proliferation.

Adenoviruses, Human↗
PubMed1996

Monitoring gene therapy with cytosine deaminase: in vitro studies using tritiated-5-fluorocytosine.

UNLABELLED: Genetically modified mammalian cells that express the cytosine deaminase (CD) gene are able to convert the nontoxic prodrug 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU). PET with 18F-5-FC may be used for in vivo measurement of CD activity in genetically modified tumors. METHODS: A human glioblastoma cell line was stably transfected with the Escherichia coli CD gene. After incubation of lysates of CD-expressing cells and control cells with 3H-5-FC high-performance liquid chromatography (HPLC) was performed. The uptake of 5-FC was measured after various incubation times using therapeutic amounts of 5-FC. In addition, saturation and competition experiments with 5-FC and 5-FU were performed. Finally, the efflux was measured. RESULTS: We found that 3H-5-FU was produced in CD-expressing cells, whereas in the control cells only 3H-5-FC was detected. Moreover, significant amounts of 5-FU were found in the medium of cultured cells, which may account for the bystander effect observed in previous experiments. However, uptake studies revealed a moderate and nonsaturable accumulation of radioactivity in the tumor cells, suggesting that 5-FC enters the cells only through diffusion. Although a significant difference in 5-FC uptake was seen between CD-positive and control cells after 48 hr of incubation, no difference was observed after 2 hr of incubation. Furthermore, a rapid efflux could be demonstrated. CONCLUSION: 5-Fluorocytosine transport may be a limiting factor for this therapeutic procedure. Quantitation with PET has to rely more on dynamic studies and modeling, including HPLC analysis of the plasma, than on nonmodeling approaches.

Chromatography, High Pressure Liquid↗
PubMed1995

Development of immunogenic colorectal cancer cell lines for vaccination: expression of CD80 (B7.1) is not sufficient to restore impaired primary T cell activation in vitro.

The capacity of colorectal carcinoma and melanoma cell lines to induce primary versus effector T lymphocyte activation in vitro was investigated. Established epithelial tumour cell lines derived from colorectal carcinoma and melanoma did not activate a primary proliferative response of resting T lymphocytes in allogeneic mixed lymphocyte tumour cell cultures (MLTCs). In contrast, the same tumour cells were effectively lysed by preactivated cytolytic T cell clones. This demonstrates that tumour cells are impaired in inducing a primary immune response but are susceptible to effector immune responses. Attempts at improving primary T cell activation revealed that exogenous cytokines, including interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and interleukin-2 (IL-2), were not effective. Expression of CD80 (B7.1), by transfecting a CD80 cDNA into the melanoma cell line SkMel63, improved T cell proliferation considerably. In contrast, CD80 expression in two colorectal carcinoma cell lines (SW480, SW707) did not result in T cell activation. This was not due to lack of class II MHC expression on SW480 since coexpression of a HLA-DR3 alloantigen and CD80 had no effect. Our data suggest that de novo CD80 expression is not, in general, sufficient to improve primary T cell activation by human tumour cells.

Antigens, Neoplasm↗
PubMed1990

[Complications of intravenous therapy with peripheral indwelling catheter].

Intravenous fluid replacement via a peripheral catheter harbours several risks. The study presented emphasizes that about 50% of the patients show complications during the first days after positioning of an intravenous line. Women patients have been affected more often than male patients. Small catheters positioned in a major vessel away from a joint in the forearm, yield the best results. Peripheral lines should be removed within 8 to 12 hours after surgical procedures, when early symptoms are observed. Complications can be avoided by following these recommendations.

Catheterization, Peripheral↗