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G Brockhoff

Publications and source records attributed to G Brockhoff.

17 recordsLinked to original sources
PubMed2006

Inhibition of TEGDMA and HEMA-induced genotoxicity and cell cycle arrest by N-acetylcysteine.

OBJECTIVES: Dental resin monomers like triethylene glycol dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA) are able to cause an imbalance of the redox state in mammalian cells. The resulting oxidative stress originating from reactive oxygen species (ROS) has been associated with cytotoxicity. We hypothesized that ROS might contribute to the generation of genotoxicity by TEGDMA and HEMA as well. Therefore, we examined the formation of micronuclei in V79 cells by both resin monomers in the presence of the antioxidant N-acetylcysteine (NAC), which scavenges ROS. In addition, we analyzed the effects of TEGDMA and HEMA on the normal cell cycle in the presence of NAC. METHODS: V79 fibroblasts were exposed to increasing concentrations of TEGDMA and HEMA in the presence and absence of NAC for 24h. Genotoxicity was indicated by the formation of micronuclei. The modification of the normal cell cycle was analyzed by flow cytometry (FACS). RESULTS: A dose-related increase in the number of micronuclei in V79 cells-induced by TEGDMA and HEMA indicated genotoxicity of both chemicals. However, the formation of micronuclei was reduced in the presence of 10 mmol/L NAC, indicating its protective role. A cell cycle delay in G2 phase caused by TEGDMA was absent when cells were co-treated with NAC. Similarly, the presence of NAC led to a reversion of the cell cycle delay in HEMA-treated cell cultures. SIGNIFICANCE: Our results suggest that genotoxic effects and the modification of the cell cycle caused by TEGDMA and HEMA are mediated, at least in part, by oxidative stress.

Acetylcysteine↗
PubMed2006

Cytomics - importance of multimodal analysis of cell function and proliferation in oncology.

Cancer is a highly complex and heterogeneous disease involving a succession of genetic changes (frequently caused or accompanied by exogenous trauma), and resulting in a molecular phenotype that in turn results in a malignant specification. The development of malignancy has been described as a multistep process involving self-sufficiency in growth signals, insensitivity to antigrowth signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and finally tissue invasion and metastasis. The quantitative analysis of networking molecules within the cells might be applied to understand native-state tissue signalling biology, complex drug actions and dysfunctional signalling in transformed cells, that is, in cancer cells. High-content and high-throughput single-cell analysis can lead to systems biology and cytomics. The application of cytomics in cancer research and diagnostics is very broad, ranging from the better understanding of the tumour cell biology to the identification of residual tumour cells after treatment, to drug discovery. The ultimate goal is to pinpoint in detail these processes on the molecular, cellular and tissue level. A comprehensive knowledge of these will require tissue analysis, which is multiplex and functional; thus, vast amounts of data are being collected from current genomic and proteomic platforms for integration and interpretation as well as for new varieties of updated cytomics technology. This overview will briefly highlight the most important aspects of this continuously developing field.

Cell Division↗
PubMed2004

Exposure to continuous bromodeoxyuridine (BrdU) differentially affects cell cycle progression of human breast and bladder cancer cell lines.

Incorporation of bromodeoxyuridine (BrdU) during DNA replication is frequently used for cell cycle analysis. The flow cytometric BrdU/Hoechst quenching technique is conducive to high-resolution assessment of cell cycle kinetics, but requires continuous BrdU treatment, which may have cytostatic or cytotoxic effects. Here, we have examined the impact of BrdU on the proliferation of BT474 and SK-BR-3 breast cancer cell lines and compared the observed effects with cell proliferation of RT4 and J82 bladder carcinoma cells, previously described to be sensitive and insensitive to BrdU, respectively. Both uni- and bi-parametric DNA measurements were performed to identify BrdU-induced alterations in the S-phase fraction and in cell cycle progression. An annexinV/propidium iodide (PI) assay was used to identify potential induction of apoptosis by BrdU. Proliferative activity in BT474, SK-BR-3, and RT4 cultures was reduced in different cell cycle phases due to continuous treatment with 60, 5.0, and 3.5 micro m BrdU. This effect, which was not found in J82 cultures, was dependent on exposure time (96 versus 48 h) and was also dose-dependent for RT4 and SK-BR-3. BrdU application does not induce apoptosis or necrosis as revealed with the annexin V/PI assay. We concluded that continuous BrdU treatment did not affect cell viability, but essentially alters cell cycle progression in three out of four cell lines tested. Cell-type specific validation of the feasibility of the powerful BrdU/Hoechst quenching technique is required and recommended.

Apoptosis↗
PubMed2004

[FISH analysis in the differential diagnosis of flat urothelial lesions using tissue microarrays].

AIMS: FISH technology offers an additional tool in the diagnosis of precancerous and malignant lesions of the urinary tract from cytological specimens. Here, we examined the relevance of chromosomal imbalance in flat urothelial lesions using FISH on paraffin-embedded tissue. In addition, the status of Her2/neu and STK15, a key molecule in the development of aneuploidy, was evaluated. METHODS: Flat lesions (normal urothelium, hyperplasia, reactive atypia, dysplasia and Carcinoma in situlCis) of 73 patients were analyzed in respect of chromosome 3, 7, 17 polysomy, deletion of p16, status of HER2/neu and of STK15 by FISH (UroVysion, PathVysion, Vysis) and immunohistochemistry (HercepTest, DAKO) using tissue microarrays. The data were correlated with histology. RESULTS AND CONCLUSIONS: Aneusomy of at least one of the chromosomes usually correlates with the histology of carcinoma in situ or invasive tumor growth. Reactive atypias, rarely showing chromosomal imbalance, can be distinguished from Cis in the majority of investigated cases, but the FISH technique is not able to differentiate reactive atypia from mild dysplasia. About 30 % of the non-neoplastic lesions like urothelial hyperplasia and normal urothelium display polysomy of at least one chromosome in more than 20% of all cells, indicating an elevated risk towards a synchronous development of a higher dysplastic lesion (i.e. Cis). Polysomy of one of three investigated chromosomes (3, 7, 17) occurs randomly within all lesions. A deletion of the p16 locus is most frequently observed in aneuploid lesions. Altered Her2/neu expression patterns are frequently observed in malignant and dysplastic lesions but also in 25 % of the non-neoplastic lesions. An overexpression of Her2/ neu is found in 10-20% of invasive urothelial carcinomas and occasionally in Cis (5 %). However, the Her2/neu gene locus is not amplified in these samples. Gene amplification of STK15 was seen in tumors as well as in normal urothelium but it is still unclear whether it indicates an elevated risk towards the development of manifest urothelial tumors.

Chromosome Aberrations↗
PubMed2003

Optimization of three- and four-color multiparameter DNA analysis in lymphoma specimens.

BACKGROUND: Simultaneous analysis of DNA and immunophenotype of lymphoma cells by flow cytometry allows the calculation of the proliferative activity and aneuploidy in even a small lymphoma population. Unfavorable DNA binding characteristics or spectral features of DNA dyes impair the accuracy of multiparameter DNA analysis and limit their clinical application. We describe here a reliable and reproducible application of both three- and four-color multiparameter DNA analysis. METHODS: After immunostaining of fresh samples of peripheral blood, bone marrow and single cell suspensions of lymph nodes from healthy and lymphoma patients, a methanol fixation for TO-PRO-3 and DRAQ5 staining was tested. RESULTS: The red-excitable TO-PRO-3 on a FACSCalibur is limited to two-color antigen staining including fluorescein-isothiocyanate and phycoerythrin-labeled monoclonal antibodies due to its broad excitation spectrum. Although DRAQ5 is only applicable to flow cytometers equipped with a single argon laser emitting 488-nm light, its emission spectrum can be easily separated from the FITC, PE, and PE/Texas-Red emissions. DRAQ5 showed almost identical stoichiometric DNA binding characteristics as propidium iodide. Coefficient of variation produced by DRAQ5 staining is in the range of 3.5 and is adequate for detecting aneuploid amd near-diploid cells. CONCLUSIONS: These advantageous features of DRAQ5 make it a reliable candidate for multiparameter clinical studies.

Anthraquinones↗
PubMed2003

[The "classical" macrophage marker CD68 is strongly expressed in primary human fibroblasts].

AIM: Monoclonal antibodies against the human homologue of mouse macrosialin, CD68, are generally commercialized as markers for human monocytes and macrophages. Indeed, CD68 is considered as a selective marker for human myeloid cells, although several previous immunohistochemical studies indicate that some antibody clones also react with other hematopoietic and non-hematopoietic cell types. The aim of our study was to verify these observations and to evaluate the reliability of CD68 as a macrophage marker. METHODS: We investigated protein and RNA expression of CD68 in various fibroblast types and carcinoma cell lines as compared to monocytes and macrophages using immunohistochemistry, flow cytometry, and specific RT-PCR. Different monoclonal antibody clones against CD68 were applied including KP-1 and EBM11. RESULTS: As expected, the intensity of immunohistochemical and flow cytometric CD68 staining was dependent on both the antibody clone and the fixation procedure. However, fibroblasts isolated from normal skin, normal breast, breast tumor tissue, and osteoarthritis synovia clearly expressed CD68 protein at levels comparable to macrophages. The specificity of CD68 expression in fibroblasts was verified by RT-PCR which also showed some tumor cell types to express CD68 mRNA. CONCLUSION: Our findings clearly demonstrate that the expression of CD68 is not restricted to the macrophage lineage. This is highly relevant for experimental and diagnostic purposes, since anti CD68 antibodies cannot be accepted without reservations for the discrimination of myeloid cells and fibroblasts even in paraffin sections after formalin fixation.

Antigens, CD↗
PubMed2001

Epidermal growth factor receptor, c-erbB2 and c-erbB3 receptor interaction, and related cell cycle kinetics of SK-BR-3 and BT474 breast carcinoma cells.

BACKGROUND: Receptors belonging to the epidermal growth factor receptor (EGFR) family transfer extracellular signals by homotypic and heterotypic receptor interaction and cross-activation. Cell differentiation, death, and proliferation are regulated via these receptor-tyrosine-kinases. However, the initial mechanisms that lead to signal specificity and diversity, which cause a defined cellular response, are incompletely understood. We investigated the recruitment of receptor complexes in two c-erbB2-overexpressing breast carcinoma cell lines, SK-BR-3 and BT474, after ligand binding and its effects on intracellular signal transduction and cell cycle regulation. METHODS: In order to analyze the coaggregation of receptors on the cell surface induced by specific growth factor treatment, we used the flow cytometric Foerster-type fluorescence resonance energy transfer (FRET) technique. Cell cycle kinetics were monitored flow cytometrically via the anti-BrdU technique and acitivation of intracellular signal cascades was analyzed by Western blotting. RESULTS: After stimulation with EGF BT474, but not SK-BR-3, cells formed EGFR/c-erbB2 receptor complexes. Neither EGF nor heregulin (HRG) induced c-erbB2/c-erbB3 receptor complexes in BT474. However, SK-BR-3 cells exhibited a high amount of c-erbB2/c-erbB3 heterodimers even without growth factor stimulation which could be elevated after prolonged EGF and HRG treatment. In both cell lines, mitogen-activated protein kinase (MAPK) phosphorylation was detectable after short-term and prolonged EGF and HRG treatment. However, only SK-BR-3 cells showed a constitutive activation of both protein kinase B (PKB)/Akt and MAPK signaling pathways. Growth factor treatment caused an amplified PKB/Akt activation in this cell line. The induction of EGFR/c-erbB2 complexes in BT474 was associated with shortening of the G1-phase of the cell cycle. In contrast, the concurrent activation of MAPK and PKB/Akt by EGF treatment led to an inhibition of proliferation in SK-BR-3 and can be attributed to missing EGFR/c-erbB2 heterodimers. HRG was a strong stimulator of proliferation in both cell lines. CONCLUSIONS: We show that in the presence of identical amounts of c-erbB2 receptors, the ligand-induced cellular response differs significantly. These differences were mediated by variances in signal transduction, most likely due to different recruitment of heterotypic receptor complexes. Overall, there is strong evidence that c-erbB2 receptor overexpression in breast cancer cells is an insufficient marker to determine cellular response in terms of cell proliferation. 2001.

Breast Neoplasms↗
PubMed2001

Flow cytometric DNA and phenotype analysis in pathology. A meeting report of a symposium at the annual conference of the German Society of Pathology, Kiel, Germany, 6-9 June 2000.

This meeting report summarizes the presentations of three different groups that are active in the field of flow cytometry (FCM) in relation to diagnosing and classification of proliferative disorders. The report starts with the contribution from Regensburg about the developments in DNA FCM, the progression to dual parameter determinations, and combination of immunophenotyping in combination with DNA. In the second part, the use of FCM for the detection of isolated tumor cells in the peripheral blood from patients with prostate or breast cancer is discussed in a contribution from Münster. In the third part, from Heerlen, the use of multi-parameter FCM on formalin-fixed paraffin-embedded tissues from solid tumors is discussed as a new development and application in routine surgical pathology.

DNA↗
PubMed2001

Development of allergic contact dermatitis requires activation of both tumor necrosis factor-receptors.

We investigated the role of the TNF receptors, type I (p55TNFR) and type II (p75TNFR), in a mouse model of contact hypersensitivity, i.e., a model of a delayed type hypersensitivity (DTH) allergic reaction. Mice deficient for p55TNFR or p75TNFR were used to investigate the functions of these receptors in development of the DTH reaction. We show that both TNF receptors have a strong influence on the overall outcome of the DTH reaction, with the two TNF receptors exerting distinct functions. Dendritic cells of mice lacking p55TNFR had a defect in allergen uptake but showed normal migration into regional lymph nodes. In contrast, dendritic cells of p75TNFR-deficient mice showed diminished migration into regional lymph nodes after allergen contact, whereas the allergen uptake was independent of the p75TNFR. Thus, both TNF receptors are required for the development of a complete DTH reaction.

Animals↗
PubMed2000

Comparative analysis of CD137 and LPS effects on monocyte activation, survival, and proliferation.

CD137 (ILA/4-1BB), a member of the TNF receptor family, regulates activation, survival and proliferation of primary human monocytes. Here we compare the activities of lipopolysaccharide (LPS), a classical and potent monocyte activator to that of CD137. LPS is a more potent activator of monocytes, as evidenced by a stronger induction of the proinflammatory cytokine IL-8. However, CD137 could further increase maximal cytokine induction by LPS, which points to separate signaling pathways for LPS and CD137. Also, expression of myc was only induced by the combination of CD137 and LPS. Expression of macrophage colony-stimulating factor is induced more potently by CD137, but an additive effect is obtained by the combination of CD137 and LPS. Monocyte/macrophage survival and proliferation is only induced by CD137. LPS counteracts both activities of CD137 via activation induced cell death. While LPS has a role in activation of monocytes in innate immunity, the CD137 receptor/ligand system seems to deliver an activating signal to monocyte in acquired immunity.

Antigens, CD↗
PubMed1999

Use of a mechanical dissociation device to improve standardization of flow cytometric cytokeratin DNA measurements of colon carcinomas.

In order to standardize dual-fluorescence DNA flow cytometry using cytokeratin (CK) antibodies, normal colonic mucosa and tumor tissue were sampled from 308 colorectal surgical specimens. Fresh colon specimens were processed directly and stored frozen until dissociation. The samples were divided into aliquots for manual dissociation with tweezers and scalpel, and parallel dissociation with an automated disaggregation device (Medimachine, DAKO Diagnostika GmbH, Hamburg, Germany). An indirect immunofluorescence method with anti-cytokeratin antibodies and propidiumiodide was applied and measured on a single-laser flow cytometer (FACScan, Becton Dickinson [BDI], Heidelberg, Germany). Evaluation with CellFit (BDI) or MultiPlus (Phoenix Flow Systems, San Diego, CA) showed that dual-parameter fluorescence propidiumiodide (DNA staining) and fluorescein-isothiocyanate (cytokeratin labeling) provides a reasonable staining method for DNA analysis of epithelial cells. No significant differences in coefficient of variation in CK-gated versus ungated cells could be observed. Normal colon mucosa served as a reliable internal, diploid DNA control. Medimachine dissociation led to a significantly higher gain of cytokeratin-positive cells compared to percentage of cytokeratin-positive cells after manual tissue disaggregation. Cytokeratin gating led to a clear-cut separation of S-phase fractions within the respective ploidy groups, irrespective of manual or automated dissociation. The S-phase fraction increased significantly from normal tissue to diploid and nondiploid tumors. In general, automated tissue preparation with the Medimachine allows simple cell-isolation for dual DNA/CK-flow cytometric measurement, improving the gain of CK-positive cells, and facilitating a standardized DNA analysis.

Carcinoma↗
PubMed1998

Aspirin suppresses the mutator phenotype associated with hereditary nonpolyposis colorectal cancer by genetic selection.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are well-known cancer preventives, which have been largely attributed to their antiproliferative and apoptosis-inducing activities. In this study, we show that microsatellite instability (MSI) in colorectal cancer cells deficient for a subset of the human mismatch repair (MMR) genes (hMLH1, hMSH2, and hMSH6), is markedly reduced during exposure to aspirin or sulindac [or Clinoril, which is chemically related to indomethacin (Indocin)]. This effect was reversible, time and concentration dependent, and appeared independent of proliferation rate and cyclooxygenase function. In contrast, the MSI phenotype of a hPMS2-deficient endometrial cancer cell line was unaffected by aspirin/sulindac. We show that the MSI reduction in the susceptible MMR-deficient cells was confined to nonapoptotic cells, whereas apoptotic cells remained unstable and were eliminated from the growing population. These results suggest that aspirin/sulindac induces a genetic selection for microsatellite stability in a subset of MMR-deficient cells and may provide an effective prophylactic therapy for hereditary nonpolyposis colorectal cancer kindreds where alteration of the hMSH2 and hMLH1 genes are associated with the majority of cancer susceptibility cases.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Evaluation of flow-cytometric three-parameter analysis for EGFR quantification and DNA assessment in human bladder carcinomas.

Flow-cytometric multi-parameter staining is an excellent method for defining tumour subpopulations. This provides further understanding of tumour heterogeneity and defines the biological relevance of tumour subpopulations. A method of quantifying the epidermal growth factor receptor (EGFR) in parallel with DNA staining, which was previously established in bladder carcinoma cell lines, was applied to twenty-five biopsies of urothelium and urothelial neoplasms. Uro5, a surface glycoprotein, was used to identify urothelial cells. Objective quantification of receptor content via flow cytometry was achieved with beads of defined numbers of antigen-binding sites, and receptor numbers obtained from urothelial and nonurothelial cells were compared with staining intensity in a three-step immunoperoxidase detection of the EGFR. The data obtained matched the immunohistochemical findings and were more sensitive in the low range (ca. 5x103) of receptors. Parallel definition of the proliferative fraction and DNA-ploidy of tumour cells means that this method satisfies the requirements of objective quantification for oncological diagnosis.

DNA, Neoplasm↗
PubMed1996

Options of flow cytometric three-colour DNA measurements to quantitate EGFR in subpopulations of human bladder cancer.

Flow cytometric multi-parameter analysis has proven to be a powerful tool to characterize subpopulations of cell suspensions, and is applied routinely in hematology. However, in studies of cancer where there is interest in defining phenotypic markers in conjunction with DNA content, this method has hardly been applied [6]. Our objective was to develop a methodology that extends previous investigations on relative and absolute quantitation of the epidermal growth factor receptor (EGFR) in dual parameter analysis in vitro on human bladder cancer cell lines [3]. In order to quantitate EGFR content in tumours and to relate it to DNA content, tumour selection, DNA-content, and EGFR-content measurements should be carried out simultaneously. Different fluorescent dyes were used to optimize DNA assessment and antibody staining, using a single laser instrument as a practical approach for clinical routine. In vitro cultures were used to validate the quality of tumour cell selection and antigen quantitation. Therefore, two urothelial tumour cell lines-lowly and highly differentiated-were incubated under different conditions: monolayer (ML), three-dimensional multi-cellular spheroids (MCS) and cocultures (COCU) with the fibroblast cell line NI were investigated and EGFR quantitation was related to S-phase fraction (SPF). Accurate determination of instrument settings allows simultaneous three-colour analysis with DNA assessment. Tumor cell selection based on staining with phycoerythrine (R-PE) against a highly expressed urothelial glycoprotein, detected with the antibody Uro5 or against cytokeratin appeared to be possible in FL2, using the fluorochrome combination fluorescein-isothiocyanate (FITC), R-PE and propidiumiodide (PI). Using this staining protocol, relative and absolute EGFR quantitation (quantum simply cellular beads) is shown to be accurate, when FITC is used for EGFR staining and measured in the green fluorescence channel (FL1). Using this colour combination EGFR content and SPF of tumour cells were compared in different growth states, and could be monitored reliably. In spite of a higher emission spectrum of 7-aminoactinomycin-D (7-AAD), this DNA stain provided no advantage over PI. Broad coefficients of variation (CV) were found when intact cells were stained, thus hindering accurate assessment of ploidy and S-phase fraction. Similarly, Syto-13, a DNA dye detected in FL1, could not be optimized for multi-parameter measurements. Although the emission maximum is at 520 nm, the spectrum is too wide to compensate fluorescence overlap in FL2 or FL3. Quantum Red (QR), used as a streptavidin conjugate in FL3, could not be combined with two other colours for DNA staining, since sufficient compensation was not obtainable when an argon ion laser is used. The coculture model allows verification of tumour cell selection and discrimination. The high differentiated tumour cell line RT4 shows an unambiguous correlation between EGFR content and S-phase fraction. The low differentiated tumour cell line J82 presents a similar pattern of post-transcriptional EGFR regulation with respect to culture condition, however, the S-phase fraction is basically unaffected.

Carcinoma↗
PubMed1994

Flow cytometric detection and quantitation of the epidermal growth factor receptor in comparison to Scatchard analysis in human bladder carcinoma cell lines.

The epidermal growth factor receptor (EGFR) is considered a tumor-related marker with potential diagnostic and prognostic value. In order to assess the sensitivity of flow cytometry to detect EGFR and to quantify receptors objectively, two human bladder carcinoma cell lines with different urothelial differentiation, RT4 and J82, were grown in vitro, and their membrane EGFR content was measured by flow cytometry. Exponential monolayers showed decrease of EGFR content after 20 min pulses with 10 ng/ml EGF in medium, as detected with the antibody EGFR1 in a double staining technique with propidium iodide for DNA evaluation. Further decrease of green fluorescence intensity was seen in cells constantly exposed to EGF. Absolute receptor numbers were determined by Scatchard analysis with radioactive EGF and resulted in relatively low receptor numbers for both cell lines (approximately 3-4 x 10(4) EGFR/cell), as well as one affinity class. These findings could be matched by absolute receptor quantification by flow cytometry, adding beads with defined antigenic sites (Quantum Simply Cellular, Microbead Corporation) to the cell suspension for staining. Our data suggest that flow cytometric EGFR detection and quantitation may be supplied to in vivo tumor samples and that measurements by multiparameter analysis may define subpopulations valuable for tumor diagnosis and judgment on tumor progression.

Biomarkers, Tumor↗