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Biomedical subjects

M Dietel

Publications and source records attributed to M Dietel.

At least 127 records · Page 7Linked to original sources
PubMed1996

Overexpression of the death-promoting gene bax-alpha which is downregulated in breast cancer restores sensitivity to different apoptotic stimuli and reduces tumor growth in SCID mice.

We have studied the expression of members of the bcl-2 family in human breast cancer. The expression pattern of these genes in breast cancer tissue samples was compared with the expression pattern in normal breast epithelium. No marked difference with regard to bcl-2 and bcl-xL expression was observed between normal breast epithelium and cancer tissue. In contrast, bax-alpha, a splice variant of bax, which promotes apoptosis, is expressed in high amounts in normal breast epithelium, whereas only weak or no expression could be detected in 39 out of 40 cancer tissue samples examined so far. Of interest, downregulation of bax-alpha was found in different histological subtypes. Furthermore, we transfected bax-alpha into breast cancer cell lines under the control of a tetracycline-dependent expression system. We were able to demonstrate for the first time that induction of bax expression in breast cancer cell lines restores sensitivity towards both serum starvation and APO-I/Fas-triggered apoptosis and significantly reduces tumor growth in SCID mice. Therefore, we propose that dysregulation of apoptosis might contribute to the pathogenesis of breast cancer at least in part due to an imbalance between members of the bcl-2 gene family.

Animals↗
PubMed1996

Molecular mechanisms and possibilities of overcoming drug resistance in gastrointestinal tumors.

Primary and acquired resistance of tumor cells to antineoplastic drugs is a major cause of the limited efficiency of chemotherapy. Gastrointestinal (GI) tumors have proven to express cytostatic drug resistance at an unusually high rate. One major reason for this is the multidrug resistant (MDR) phenotype which is often found in carcinomas of the stomach, bile duct, pancreas, liver, and colon. MDR is due to the overexpression of a membrane-bound glycoprotein, the so called P-glycoprotein. However, this is not the only resistance mechanisms of GI tumor cells, but the intracellular compartmentalization of drugs with subsequent release to the microenvironment represents an additional potent mechanism of drug resistance. This is independent of P-glycoprotein and as yet cannot be reversed. Alterations of glutathione-S-transferase (GST) and topoisomerase I and II may be involved either. Analyses of cell lines for cross resistance against a battery of cytostatic drugs suggest even more mechanisms which may contribute to the marked resistance of gastrointestinal cancer. Only a detailed investigation of all different types of drug insensitivity, if ever possible, might offer a chance to fully understand this multifactorial orchestra of events and to develop complex strategies for overcoming drug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

[Comparative genomic hybridization. A screening method in genetic tumor diagnosis].

Comparative genomic hybridization (CGH) is a new method of screening a tumor for genetic changes. The alterations are classified as DNA gains and losses and reveal a characteristic pattern that includes mutations at the chromosomal and subchromosomal levels. Although DNA from fresh-frozen tissue is recommended as the starting material, archival specimens can also be analyzed. Various examples are presented that illustrate the changes that occur in different tumor entities, and preneoplastic lesions, the differentiation of primary tumors from metastases, and the investigation of tumor cell lines that are resistant to cytostatic drugs. These examples emphasize that CGH can extend the possibilities for genetic diagnosis in tumor pathology.

Cell Line↗
PubMed1996

[Analysis program for quantitative detection of chromosome aberrations using comparative genomic hybridization].

Comparative genomic hybridization (CGH) is a molecular cytogenetic method for the detection of chromosomal imbalances between a tumor and a normal genome. In order to produce quantitative and reproducible results, we developed an image analysis program that allows the detection and mapping of the genetic alterations. The result is represented as a CGH sum karyogram in which the genetic changes are documented as color coded-chromosomes. The aim of our investigation is to correlate the genotype with the phenotype on the basis of CGH sum karyograms and thus to achieve a genetic characterization that will supplement the morphological tumor description.

Cell Transformation, Neoplastic↗
PubMed1996

Subcutaneous crystal-storing histiocytosis associated with lymphoplasmacytic lymphoma (immunocytoma)

A case of massive crystal-storing histiocytosis with extensive involvement of the subcutaneous adipose tissue in a 61-year-old caucasian woman with a lymphoplasmacytic lymphoma (immunocytoma) in transformation to a large cell lymphoma is presented. Paraffin-section immunohistochemistry demonstrated a monoclonal IgM/ kappa immunphenotype of the lymphoma cells and revealed IgM/kappa and, to a lesser extent, IgG/lambda in the crystal-containing histiocytes. Ultrastructurally, the electron dense intracytoplasmic crystals had variables shapes, were occasionally membrane-bound and measured up to 6 microns. The finding are briefly discussed.

Crystallization↗
PubMed1996

[Allele losses in squamous cell carcinomas of the larynx].

BACKGROUND: Loss of function of tumor suppressor genes is important in the origin and progression of malignant tumors. Analysis for loss of heterozygosity (LOH) in tumors has been used to detect chromosomal regions that could harbor these genes. METHODS: We investigated 30 paired samples of tumor and normal DNA of 20 patients with laryngeal squamous cell carcinoma. Using polymerase chain reaction (PCR), we studied microsatellite polymorphisms on 3 p, 5q, 9 p, 9q, and 17 p. We separated PCR products by denaturating gel electrophoresis and then visualized them colorimetrically. RESULTS: Allelic loss was observed most frequently on 3 p (45%), followed by 9 p (15%), 17 p (6%), and 5 q (5%), while chromosome 9 q displayed no LOH. When primary tumor and lymph node metastases were investigated, the same genetic pattern was observed. No correlation was found between LOH and tumor stage. The commonly deleted region of 3 p was at the chromosomal bands 3 p24-3 p25. Our data demonstrate that chromosome 3 p allelic loss is a common event in head and neck cancers and suggest that tumor suppressor genes on chromosome 3 p contribute to the pathogenesis of these tumors. CONCLUSIONS: A longer follow-up will reveal if the assessment of LOH can be associated with prognostic significance in laryngeal carcinomas.

Aged↗
PubMed1996

[Genetic screening of head-neck carcinomas using comparative genomic hybridization (CGH)].

BACKGROUND: Comparative Genomic Hybridization (CGH) is a novel cytogenetic method that allows the comprehensive analysis of a tumor genome for DNA gains and losses. METHODS: CGH was performed on genomic DNA extracted from 14 primary head and neck squamous cell carcinomas. Equal amounts of biotin-labeled tumor DNA and digoxigenin-labeled normal reference DNA were hybridized to normal metaphase chromosomes. The tumor DNA was visualized with fluorescein (FITC) and the normal DNA with rhodamine (TRITC) and detected in a fluorescence microscope. The signal intensities of the different fluorochromes were quantitated as gray levels along the single chromosomes. The over-and underrepresented DNA segments were quantified by computation of FITC/TRITC ratio images and average ratio profiles. RESULTS: Consensus deletion regions were most frequently observed on chromosome arms 3p (14 cases), 9p (11), 13q (10), 18q (10), 5q (9), 4q (9), 4p (7), 11q (7), 6q (6), 8p (6), and 11p (6). Copy number increases were identified for chromosomes 3q (11), 16p (9), 17q (9), 19p (8), 19q (7), 22 (7), 1p (6), 8q (6), 9q (6), and 20q (6). Particularly, the 3q isochromosome formation (3p loss/3q amplification), found in 10 cases, was a basic alteration. In addition, 11 tumors showed a 11 q13 amplification. CONCLUSION: CGH analysis allows the identification of recurrent genetic alterations in head and neck squamous cell carcinomas that will be associated with specific tumor phenotypes like metastatic behavior and prognosis.

Adult↗
PubMed1996

Immunohistochemical differentiation of metastatic breast carcinomas from metastatic adenocarcinomas of other common primary sites.

To test the possibility of immunohistochemical differentiation of cytostatically treatable metastatic breast carcinomas from other metastatic adenocarcinomas of unknown primary site, we studied a total of 328 metastatic adenocarcinomas including 35 bronchogenic, 26 pancreatic, 25 colonic, 39 gastric, 45 renal, 29 ovarian and 129 breast carcinomas with a panel of 13 commercially available monoclonal antibodies. The expression of gross cystic disease fluid protein 15 and/or oestrogen or progesterone receptors had a sensitivity of 0.83, a specificity of 0.93 and a predictive accuracy of 0.92 for carcinomas of the breast against all other carcinomas. Excluding ovarian carcinomas, this combination had a sensitivity, specificity and predictive accuracy for mammary carcinomas of 0.83, 0.98 and 0.98, respectively. Carcinoembryonic antigen and/or cytokeratin 20 identified bronchogenic, gastric, pancreatic and colorectal carcinomas versus breast carcinomas lacking gross cystic disease fluid protein 15 and oestrogen or progesterone receptors with a sensitivity, specificity and predictive accuracy of 0.82, 0.99 and 0.95, respectively. Vimentin differentiates renal cell carcinomas from gross cystic disease fluid protein 15 and oestrogene or progesterone receptor negative breast carcinomas with a sensitivity, specificity and predictive accuracy of 0.93, 0.82 and 0.84. Thus, it should be possible to differentiate most metastatic mammary carcinomas from metastatic adenocarcinomas of other common primary sites, even if the former lack expression of gross cystic disease fluid protein 15 and oestrogen or progesterone receptors.

Adenocarcinoma↗
PubMed1996

Use of non-radioactive detection in SSCP, direct DNA sequencing and LOH analysis.

Aims-To develop a protocol that is applicable to single strand conformation polymorphism (SSCP), direct sequencing and loss of heterozygosity analysis of DNA.Methods-The protocol is based on the detection of biotinylated DNA by a Streptavidin-alkaline phosphatase conjugate. Biotinylation of DNA was achieved by using 5'-end biotinylated primers for PCR. After polyacrylamide gel electrophoresis, the DNA fragments were transferred to a nylon membrane by contact blotting. Depending on the alkaline phosphatase substrate, DNA was visualised either colorimetrically or by chemiluminescence.Results-The method was verified by the identification and characterisation of p53 mutations by SSCP analysis and direct DNA sequencing, as well as the assessment of DNA loss in human lung carcinomas by microsatellite polymorphism allelotyping.Conclusions-The protocol is simple, does not require specialised equipment and would be particularly useful for laboratories with experience in Streptavidin-biotin methodology.

Journal Article↗
PubMed1996

[Morphological and functional features of cytostatic drug resistance and the effects of MDR modulators].

Manifold mechanisms of resistance can be expressed by malignancies. Profound information on this aspect is a prerequisite for comprehensive individual chemotherapy. Based on both morphological and functional findings, the diagnosis of P-Glycoprotein (P-Gp) mediated Multidrug Resistance (MDR) can be verified. In order to describe minimum criteria for conclusive diagnosis, morphological and biochemical findings (Immunocytochemistry, RT-PCR, ultrastructural and Laser-Scan microscopy) and functional data (cytostatic drug transport, proliferation) were correlated in tumor cell lines of the MDR phenotype as opposed to cells with atypical resistance. Frequently, single features of MDR are found in atypical, P-Gp negative resistance. Accumulation deficits for mitoxantrone based on vesicular drug transport were found in P-Gp negative gastric carcinoma cell line EPG85-257RNOV. Nocodazole blocked microtubule formation which is essential for vesicle transfer from perinuclear regions to the periphery of the cytosol. Cytochalasin blocked exocytosis of drug containing vesicles. MDR modulators were ineffective. Alternatively, P-Gp mediated drug extrusion and exocytosis of drug containing vesicles may constitute complementary mechanisms of resistance. In gastric carcinoma cells EPG85-257DAU, MDR modulators do increase cytosolic daunorubicin load, but drug binding to nuclear target sites is still inhibited due to drug containment within vesicles. To complicate matters, MDR modulators may be functional even in MDR negative cells, as shown in a panel of melanoma cell lines. Data show that conclusive diagnosis of P-Gp-mediated MDR should be based on more than one experimental approach including immunocytochemistry, a sensitive assay such as RT-PCR and--whenever feasible--a functional test.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

In vivo reversibility of multidrug resistance by the MDR-modulator dexniguldipine (niguldipine derivative B859-35) and by verapamil.

The newly synthesized dihydropyridine derivative B859-35 was previously shown in vitro to be highly effective in reversing multidrug resistance (MDR) of P-glycoprotein positive tumor cell lines, such as the adriamycin (ADR) resistant erythroleukemia F4-6RADR cells. In the current study B859-35 was investigated for its efficiency in reversing MDR in an in vivo tumor model for preclinical testing of MDR-modulators. F4-6RADR cells were injected into the right flank of nude mice while the parent cells were injected into the left flank. The animals were treated i.p. with ADR (9.0 mg/kg body weight) combined with B859-35 (5, 10, or 25 mg/kg) or, for comparison and validation, with verapamil (VRP) (75 mg/kg). The effects of ADR and the MDR-modulator combination were evaluated by histological morphometry of the tumors. While ADR alone was shown to be ineffective in resistant cells, the combinations of ADR + B859-35 as well as of ADR + VRP were highly active in reducing the number of viable cells in the resistant tumor nodule by 67 +/- 9% or by 53 +/- 11% of controls. This model provides evidence that even in vivo, MDR modulators can be effective in reversing drug resistance. In addition, it presents a potentially useful and rapid preclinical system for in vivo studies on the modification of drug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Similar cleavage efficiencies of an oligoribonucleotide substrate and an mdr1 mRNA segment by a hammerhead ribozyme.

Hammerhead ribozymes (Rz) can specifically recognize and cleave target RNAs in trans. This makes them attractive in antisense RNA approaches for specific gene inactivation in vivo. A severe limitation is the poor cleavage efficiency of large RNA substrates, in contrast to the high activities observed with small oligoribonucleotides (oligos) as model substrates. It was suggested that the low efficiency is caused by poor accessibility of the target sequence in the structure of the long RNA substrates. This means it should be possible to overcome this limitation by judicious choice of the target sequence, although experimental proof was lacking. We observed similar cleavage efficiencies of small and large RNA substrates with a hammerhead Rz directed against multidrug resistance-encoding mdr1 mRNA. Accordingly, large RNAs can also be good substrates, if an optimal target sequence is selected.

Base Sequence↗
PubMed1995

c-erbB-2 anti-sense phosphorothioate oligodeoxynucleotides inhibit growth and serum-induced cell spreading of P185c-erbB-2-overexpressing ovarian carcinoma cells.

Overexpression of the c-erbB-2 proto-oncogene product (p185c-erbB-2) occurs frequently in different types of human cancer and is correlated with a significantly decreased survival in ovarian cancer patients. The effect of c-erbB-2 anti-sense phosphorothioate oligodeoxynucleotides (S-ODNs) was examined on the ovarian cancer cell line SK-OV-3. p185c-erbB-2 levels were specifically reduced by a single-dose application of 5 microM c-erbB-2 anti-sense S-ODNs. This was accompanied by a 60% inhibition of anchorage-dependent cell growth. More strikingly, c-erbB-2 anti-sense S-ODNs almost completely abrogated serum-induced cell spreading. A control of complementary sense oligodeoxynucleotides did not show significant inhibitory effects on cell growth or on cell spreading. The inhibition of cell spreading was imitated by a monoclonal antibody (9G6) targeting the extracellular domain of p185c-erbB-2 and by the tyrosine kinase inhibitor erbstatin. The inhibitory activity of these 2 compounds was lost after a few hours, while the inhibition of serum-induced cell spreading by anti-sense S-ODNs was still present after 24 hr. Our results show that c-erbB-2 anti-sense S-ODNs effectively inhibit the mitogenic and spreading activity of p185c-erbB-2 in ovarian cancer cells. Thus, anti-sense strategies have the potential of providing new strategies for the therapy of ovarian cancer.

Antineoplastic Agents↗
PubMed1995

Intracellular localization, vesicular accumulation and kinetics of daunorubicin in sensitive and multidrug-resistant gastric carcinoma EPG85-257 cells.

In the human gastric carcinoma cell line EPG85-257P (parent) induction of resistance to daunorubicin (DAU) was achieved by selection with stepwise increased concentrations of the drug. The new variant was named EPG85-257DAU and was shown to overexpress the mdr1 gene product 170 kDa P-glycoprotein (P-Gp) as demonstrated by immunocytochemistry and mdr1-specific RT-PCR. To investigate the intracellular pathway of DAU the subcellular distribution of this autofluorescent drug was studied in the resistant cells and compared to its chemosensitive counterpart EPG85-257P. When sensitive cells were exposed to DAU the drug rapidly accumulated in the nucleus until cell death. No redistribution of DAU to the cytoplasm was observed. In resistant cells exposed to the drug DAU also accumulated in the nucleus but to a lesser extent than in parent cells. Following exposure, nuclear fluorescence was observed to decrease over a time period of up to 48 h. Six hours after DAU exposure formation of fluorescent vesicle formation started in the perinuclear region and increased continuously. After 48 h nuclear fluorescence was no longer detectable and DAU was located exclusively in vesicles. During this period the vesicles moved from the region of origin to the cell periphery. A pulse chase experiment showed, that vesicles may contain DAU derived from the nucleus. Treatment of EPG85-257DAU cells with DAU in conjunction with the chemosensitizer cyclosporin A (CsA) increased nuclear fluorescence without impairing vesicle formation. Disruption of microtubules by nocodazole led to an accumulation of vesicles in the perinuclear region indicating that microtubules are involved in vesicular transport. Treatment of EPG85-257DAU cells with the actin disruptor cytochalasin B led to accumulation of vesicles in the cell periphery indicating that actin may be involved in exocytosis. Uptake and efflux of DAU and rhodamin (RH) were determined in sensitive and resistant cells using a fluorescence activated cell sorter. Uptake of both compounds was distinctly lower in resistant than in sensitive cells. When resistant cells preloaded for 2 h with RH subsequently were incubated in drug free medium the substance was rapidly released indicating transmembrane transport by P-Gp. In contrast, despite expression of P-Gp in resistant cells no considerable release of DAU was observed for up to 2 h under the same experimental protocol. This indicates that in resistant cells intracellular DAU at least in part may be inaccessible for P-Gp and that vesicular drug transport appears to contribute to DAU resistance by removing intracellular DAU via exocytosis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Secondary combined resistance to the multidrug-resistance-reversing activity of cyclosporin A in the cell line F4-6RADR-CsA.

Multidrug-resistant tumor cells can be resensitized by combined application of the selecting cytostatic drug and a chemosensitizer, such as cyclosporin A (CsA) or a calcium channel blocker. Since clinical trials on the circumvention of multidrug resistance (MDR) with chemosensitizers report disparate results, we investigated whether tumor cells of the MDR phenotype can develop additional resistance to the cytostatic chemosensitizer combination. Thus, the Adriamycin(ADR)-selected, P-glycoprotein-positive MDR Friend leukemia cell line F4-6RADR was exposed to stepwise increased concentrations of CsA at a constant level of 0.05 microgram/ml ADR. The initial CsA concentration (plus 0.05 microgram/ml ADR) to inhibit cell growth of F4-6RADR cells by 50% (IC50) was 0.04 microgram/ml. By continuous incubation for more than 6 months, the IC50 for CsA (at constant ADR) was elevated to 3.6 micrograms/ml (90-fold), thus generating the variant F4-6RADR-CsA. The F4-6RADR-CsA cells were cross-resistant for cyclosporin H (CsH), a non-immunosuppressive derivative of CsA. As shown by immunocytochemistry as well as by the polymerase chain reaction and by Western blotting including densitometry, P-glycoprotein was preserved in the F4-6RADR-CsA variant and was expressed at a 4-fold higher level than in F4-6RADR cells. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis could detect no new proteins in F4-6RADR-CsA as compared to F4-6RADR. Interestingly, resistance of F4-6RADR-CsA cells remained reversible for the calcium antagonists verapamil and dihydropyridine B859-35 (dexniguldipine-HCl), indicating that CsA and these compounds interfere with the P glycoprotein function by different pharmacodynamic mechanisms. Transport studies with [14C]ADR, performed in the presence and absence of chemosensitizers, confirmed the good correlation of P-glycoprotein function with the pattern of resistance found in proliferation assays. Cellular accumulation of [3H]cyclosporin was reduced to 71% of that of the F4-6 controls in F4-6RADR-CsA cells, but remained at the level of controls in F4-6RADR cells. Results indicate that increased amounts of the P-glycoprotein--besides other, perhaps more important mechanisms that are as yet unknown--partially mediate CsA resistance in F4-6RADR-CsA cells. We have designated this new form of resistance "secondary combined resistance" (SCR). The results suggest that at least some clinical cases of insensitivity to chemosensitizers or of relapse after reversing therapy could be explained by SCR, and that resensitizing treatment of tumor patients should be based on the consideration of several chemosensitizers of different pharmacodynamics.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Reversion of multidrug resistance in the P-glycoprotein-positive human pancreatic cell line (EPP85-181RDB) by introduction of a hammerhead ribozyme.

A major problem in cytostatic treatment of many tumours is the development of multidrug resistance (MDR4). This is most often accompanied by the overexpression of a membrane transport protein, P-glycoprotein, and its encoding mRNA. In order to reverse the resistant phenotype in cell cultures, we constructed a specific hammerhead ribozyme possessing catalytic activity that cleaves the 3'-end of the GUC sequence in codon 880 of the mdr1 mRNA. We demonstrated that the constructed ribozyme is able to cleave a reduced substrate mdr1 mRNA at the GUC position under physiological conditions in a cell-free system. A DNA sequence encoding the ribozyme gene was then incorporated into a mammalian expression vector (pH beta APr-1 neo) and transfected into the human pancreatic carcinoma cell line EPP85-181RDB, which is resistant to daunorubicin and expresses the MDR phenotype. The expressed ribozyme decreased the level of mdr1 mRNA expression, inhibited the formation of P-glycoprotein and reduced the cell's resistance to daunorubicin dramatically; this means that the resistant cells were 1,600-fold more resistant than the parental cell line (EPP85-181P), whereas those cell clones that showed ribozyme expression were only 5.3-fold more resistant than the parental cell line.

ATP Binding Cassette Transporter, Subfamily B, Mem↗