Franz Thiebaut - Academia.edu (original) (raw)
Papers by Franz Thiebaut
Scanning electron microscopy, 1984
7,12 dimethylbenz-a-anthracene (DMBA)-treated C3H/10T-1/2 mouse embryo fibroblasts have been char... more 7,12 dimethylbenz-a-anthracene (DMBA)-treated C3H/10T-1/2 mouse embryo fibroblasts have been characterized by the occurrence of pleomorphic microvilli (PMV) in contrast to the non-treated smooth-surfaced C3H/10T-1/2 cells. Recent studies by semiquantitative secondary electron image (SEI) have demonstrated a significant correlation between the number of cells with numerous PMV and the development of oncogenic potential. In these studies, so-called type III transformants scored highest in oncogenic potential and occurrence of cells with pleomorphic microvilli, but always contained a small fraction of cells displaying rather smooth surfaces. Quantitation of the size of nuclei and nucleoli and the number of intranucleolar silver granules after nucleolar organizer region (AgNOR) staining and back scattered electron image (BEI) with an automatic image analysis system (IBAS) revealed that these smooth-surfaced type III cells were significantly different (p less than 0.001) from the non-DMB...
Journal of Histochemistry & Cytochemistry, 1989
Using peroxidase immunohistochemistry, we examined the distribution of P170, a multidrug transpor... more Using peroxidase immunohistochemistry, we examined the distribution of P170, a multidrug transport protein, in normal tissues by use of two different monoclonal antibodies (MAb). MAb MRK16 is a MAb that has been shown to react with an epitope in P170 located on the external face of the plasma membrane of multidrug-resistant human cells. MAb C219 has been shown to react with P170 in many mammalian species, and detects an epitope located on the cytoplasmic face of the plasma membrane. Using MRK16, we have previously described the localization of P170 on the bile canalicular face of hepatocytes, the apical surface of proximal tubular cells in kidney, and the surface epithelium in the lower GI tract in normal human tissues. In this work, we report that MRK16 also detects P170 in the capillaries of some human brain samples. A similar pattern was found using MAb C219 in rat tissues. in addition, MAb C219 showed intense localization in selected skeletal muscle fibers and all cardiac muscle...
We have previously shown that forskolin and 3-isobutyl-1-methylxanthine (IBMX) increased accumula... more We have previously shown that forskolin and 3-isobutyl-1-methylxanthine (IBMX) increased accumulation of cisplatin (DDP) in DDP-sensitive 2008 human ovarian carcinoma cells in proportion to their ability to increase cAMP. Since the major function ofcAMP is to activate protein kinase A, it was conjec-tured that the stimulation of DDP accumulation was mediated by a protein kinase A substrate. We now show that exposure of 2008 cells to forskolin resulted in phosphorylation of a promi-nent 52-kD membrane protein. Microsequencing of the band demonstrated it to be human j-tubulin. Similarly, pretreatment of 2008 cells with the microtubule stabilizing drug taxol in-creased platinum accumulation in a dose-dependent manner. In 11-fold DDP-resistant 2008/C13 * 5.25 cells, decreased DDP accumulation was associated with enhanced spontaneous for-mation of microtubule bundles and decreased expression of 6-tubulin and the tubulin-associated p53 antioncogene relative to 2008 cells. 2008/C13 * 5.25 ...
Journal of Histochemistry & Cytochemistry, 1990
Multidrug-resistant cells contain a plasma membrane efflux pump, the multidrug transporter, which... more Multidrug-resistant cells contain a plasma membrane efflux pump, the multidrug transporter, which actively expels certain hydrophobic drugs from the cytosol to the cell exterior. These drugs are usually positively charged at physiological pH. Because one might predict that this efflux of positively charged molecules might deplete the cytosol of protons, raising the cytosolic pH, we examined the cytosolic pH of multidrug-resistant cells directly using a pH-sensitive dye coupled to a membrane-impermeable molecule. The dye (SNARF), covalently coupled to 10,000 MW dextran, was mechanically microinjected into the cytosol of cultured multidrug-resistant mouse NIH3T3 cells which express the human multidrug transporter. The fluorescence emission of the dye in living cells was measured using epifluorescence microscopy at different wavelengths to provide a measure of the pH of the cytosolic environment. Multidrug-resistant cells had a higher cytosolic pH than drug-sensitive normal parental ce...
Chromosoma, 1985
By using simultaneously the AgNOR silver staining method, back-scattered electron imaging mode an... more By using simultaneously the AgNOR silver staining method, back-scattered electron imaging mode and stereo-tilt in scanning electron microscopy (SEM), it is possible to observe the nucleus through the cell surface, the nucleolus, and the tri-dimensional distribution of the Ag-NOR-associated acidic proteins. In C3H10T1:2 cells and their 7-12-dimethylbenz-alpha-anthracene-treated transformants, the staining demonstrates several intranucleolar silver-staining granules (SSG), surrounded by a weakly staining region. The SSG may represent the fibrillar center (FC) and the weakly staining region, the fibrillar dense component (FD). This component can link several SSG together to form a "rope-like structure". In cells with no visible nucleolus and inactive nucleolar organizer regions (NORs) the silver-staining granules are less numerous, close together and the presumed fibrillar dense components are not visible. The SSG are located more peripherally, and the weakly staining region and the "rope-like structure" are less prominent in control cell nucleoli than in transformed cells with a comparatively high rate of RNA synthesis.
International Journal of Cancer, 1991
We isolated an IgG2a murine monoclonal antibody (MAb) termed MAb57, specifically reactive with mu... more We isolated an IgG2a murine monoclonal antibody (MAb) termed MAb57, specifically reactive with multi-drug-resistant (MDR) human cells. Its specificity toward the MDRI gene product (P-glycoprotein) has been demonstrated by the concordant segregation of the MAb57 epitope with the MDRI gene in interspecific mouse x human cell hybrids, and the reactivity of several different MDRI gene-expressing cells with MAb57, particularly insect cells acutely infected with a baculovirus encoding the MDRI gene. MAb57 can be used to detect, by flow cytometry, variations in the relative drug-resistance levels of several MDR KB and CEM cell variants. This immunological probe has also proven useful in selectively destroying MDR target cells in an antibody-dependent cell-mediated (ADCC) assay system as well as in detecting P-glycoprotein expression in normal and malignant tissues and cells.
Stain Technology, Jun 1, 1984
Some recently developed silver staining methods allow selective staining of acidic nucleolar prot... more Some recently developed silver staining methods allow selective staining of acidic nucleolar proteins. Pretreating deparaffinized sections with Schiff's reagent improves the specificity of Goodpasture and Bloom's AgNOR staining (as modified by Kodama et al.) after aldehyde fixation.
Acta Histochemica Et Cytochemica, 1987
FEBS Letters, 1993
Multidrug-resistant KB-V1 cells carry amplified mdrl gene sequences located in an extrachromosoma... more Multidrug-resistant KB-V1 cells carry amplified mdrl gene sequences located in an extrachromosomal compartment (on episomes). Since episomes do not contain centromeric or telomeric sequences it is unclear whether they are able to bind to nuclear matrix proteins that may regulate episomal gene expression. Using high salt treatments followed by in situ hybridization and dot blot analyses we found evidence for direct binding of episomal DNA to nuclear matrix proteins. This binding could only be reversed after incubation with trypsin or proteinase K as determined by contour-clamped homogeneous electric field (CHEF) electrophoresis. Our findings are consistent with the concept that circular extrachromosomal DNA may not only reintegrate into nuclear DNA but may also be subject to functional control by regulatory proteins within the nuclear matrix.
A method for the observation of junctional complexes by backscattered electron imaging in scannin... more A method for the observation of junctional complexes by backscattered electron imaging in scanning electron microscopy, in tissue blocks, is presented. The junctional complexes are revealed by a modified silver staining method (originally devised for nucleolar organizer regions), used "en bloc" after formalin or glutaraldehyde fixation. Backscattered electron imaging allows, after this staining, the observation of junctional complexes through the surface of intact superficial cells, in the three tissues studied (liver, jejunum and urinary bladder). The interest of this approach is to offer the possibility of observing junctional complexes "by transparence," in nondissociated and nonsectioned tissues.
Proceedings of the National Academy of Sciences of the United States of America, Nov 1, 1987
Monoclonal antibody MRK16 was used to determine the location of P-glycoprotein, the product of th... more Monoclonal antibody MRK16 was used to determine the location of P-glycoprotein, the product of the multidrug-resistance gene (MDR1), in normal human tissues. The protein was found to be concentrated in a small number of specific sites. Most tissues examined revealed very little P-glycoprotein. However, certain cell types in liver, pancreas, kidney, colon, and jejunum showed specific localization of P-glycoprotein. In liver, P-glycoprotein was found exclusively on the biliary canalicular front of hepatocytes and on the apical surface of epithelial cells in small biliary ductules. In pancreas, P-glycoprotein was found on the apical surface of the epithelial cells of small ductules but not larger pancreatic ducts. In kidney, P-glycoprotein was found concentrated on the apical surface of epithelial cells of the proximal tubules. Colon and jejunum both showed high levels of P-glycoprotein on the apical surfaces of superficial columnar epithelial cells. Adrenal gland showed high levels of P-glycoprotein diffusely distributed on the surface of cells in both the cortex and medulla. These results suggest that the protein has a role in the normal secretion of metabolites and certain anti-cancer drugs into bile, urine, and directly into the lumen of the gastrointestinal tract.
Molecular and Cellular Biology of Multidrug Resistance in Tumor Cells, 1991
Cancer research, 1991
The effect of expression of the c-Ha-ras oncogene on cisplatin (DDP) sensitivity was examined in ... more The effect of expression of the c-Ha-ras oncogene on cisplatin (DDP) sensitivity was examined in murine NIH 3T3 cells transfected with the dexamethasone (DEX)-inducible mouse mammary tumor virus promoter linked to an activated c-Ha-ras gene [LTR H-ras(A) cells]. Treatment of these cells with 5 microM DEX for 24 h induced c-Ha-ras expression and produced an 8.2 +/- 1.3-fold (SD) increase in DDP resistance as quantitated by clonogenic assay. Induction of the c-Ha-ras oncogene reduced DDP accumulation by 40% and intrastrand adduct formation by 17%. In nontransfected wild-type NIH 3T3 cells, DEX did not induce DDP resistance nor did it decrease DDP accumulation. Induction of c-Ha-ras expression did not alter cellular glutathione content or the activity of glutathione-S-transferase in the LTR H-ras(A) cells. DEX increased cellular metallothionein content by 1.6-fold in NIH 3T3 cells and 3.3-fold in LTR H-ras(A) cells. We conclude that DEX-induced overexpression of a mutant c-Ha-ras gene ...
Platinum and Other Metal Coordination Compounds in Cancer Chemotherapy 2, 1996
Correlative Microscopy in Biology, 1987
Proceedings of the National Academy of Sciences, 1987
Monoclonal antibody MRK16 was used to determine the location of P-glycoprotein, the product of th... more Monoclonal antibody MRK16 was used to determine the location of P-glycoprotein, the product of the multidrug-resistance gene (MDRJ), in normal human tissues. The protein was found to be concentrated in a small number of specific sites. Most tissues examined revealed very little Pglycoprotein. However, certain cell types in liver, pancreas, kidney, colon, and jejunum showed specific localization of P-glycoprotein. In liver, P-glycoprotein was found exclusively on the biliary canalicular front of hepatocytes and on the apical surface of epithelial cells in small biliary ductules. In pancreas, P-glycoprotein was found on the apical surface of the epithelial cells of small ductules but not larger pancreatic ducts. In kidney, P-glycoprotein was found concentrated on the apical surface of epithelial cells of the proximal tubules. Colon and jejunum both showed high levels ofP-glycoprotein on the apical surfaces of superficial columnar epithelial cells. Adrenal gland showed high levels of P-glycoprotein diffusely distributed on the surface ofcells in both the cortex and medulla. These results suggest that the protein has a role in the normal secretion of metabolites and certain anti-cancer drugs into bile, urine, and directly into the lumen of the gastrointestinal tract.
Scanning electron microscopy, 1984
7,12 dimethylbenz-a-anthracene (DMBA)-treated C3H/10T-1/2 mouse embryo fibroblasts have been char... more 7,12 dimethylbenz-a-anthracene (DMBA)-treated C3H/10T-1/2 mouse embryo fibroblasts have been characterized by the occurrence of pleomorphic microvilli (PMV) in contrast to the non-treated smooth-surfaced C3H/10T-1/2 cells. Recent studies by semiquantitative secondary electron image (SEI) have demonstrated a significant correlation between the number of cells with numerous PMV and the development of oncogenic potential. In these studies, so-called type III transformants scored highest in oncogenic potential and occurrence of cells with pleomorphic microvilli, but always contained a small fraction of cells displaying rather smooth surfaces. Quantitation of the size of nuclei and nucleoli and the number of intranucleolar silver granules after nucleolar organizer region (AgNOR) staining and back scattered electron image (BEI) with an automatic image analysis system (IBAS) revealed that these smooth-surfaced type III cells were significantly different (p less than 0.001) from the non-DMB...
Journal of Histochemistry & Cytochemistry, 1989
Using peroxidase immunohistochemistry, we examined the distribution of P170, a multidrug transpor... more Using peroxidase immunohistochemistry, we examined the distribution of P170, a multidrug transport protein, in normal tissues by use of two different monoclonal antibodies (MAb). MAb MRK16 is a MAb that has been shown to react with an epitope in P170 located on the external face of the plasma membrane of multidrug-resistant human cells. MAb C219 has been shown to react with P170 in many mammalian species, and detects an epitope located on the cytoplasmic face of the plasma membrane. Using MRK16, we have previously described the localization of P170 on the bile canalicular face of hepatocytes, the apical surface of proximal tubular cells in kidney, and the surface epithelium in the lower GI tract in normal human tissues. In this work, we report that MRK16 also detects P170 in the capillaries of some human brain samples. A similar pattern was found using MAb C219 in rat tissues. in addition, MAb C219 showed intense localization in selected skeletal muscle fibers and all cardiac muscle...
We have previously shown that forskolin and 3-isobutyl-1-methylxanthine (IBMX) increased accumula... more We have previously shown that forskolin and 3-isobutyl-1-methylxanthine (IBMX) increased accumulation of cisplatin (DDP) in DDP-sensitive 2008 human ovarian carcinoma cells in proportion to their ability to increase cAMP. Since the major function ofcAMP is to activate protein kinase A, it was conjec-tured that the stimulation of DDP accumulation was mediated by a protein kinase A substrate. We now show that exposure of 2008 cells to forskolin resulted in phosphorylation of a promi-nent 52-kD membrane protein. Microsequencing of the band demonstrated it to be human j-tubulin. Similarly, pretreatment of 2008 cells with the microtubule stabilizing drug taxol in-creased platinum accumulation in a dose-dependent manner. In 11-fold DDP-resistant 2008/C13 * 5.25 cells, decreased DDP accumulation was associated with enhanced spontaneous for-mation of microtubule bundles and decreased expression of 6-tubulin and the tubulin-associated p53 antioncogene relative to 2008 cells. 2008/C13 * 5.25 ...
Journal of Histochemistry & Cytochemistry, 1990
Multidrug-resistant cells contain a plasma membrane efflux pump, the multidrug transporter, which... more Multidrug-resistant cells contain a plasma membrane efflux pump, the multidrug transporter, which actively expels certain hydrophobic drugs from the cytosol to the cell exterior. These drugs are usually positively charged at physiological pH. Because one might predict that this efflux of positively charged molecules might deplete the cytosol of protons, raising the cytosolic pH, we examined the cytosolic pH of multidrug-resistant cells directly using a pH-sensitive dye coupled to a membrane-impermeable molecule. The dye (SNARF), covalently coupled to 10,000 MW dextran, was mechanically microinjected into the cytosol of cultured multidrug-resistant mouse NIH3T3 cells which express the human multidrug transporter. The fluorescence emission of the dye in living cells was measured using epifluorescence microscopy at different wavelengths to provide a measure of the pH of the cytosolic environment. Multidrug-resistant cells had a higher cytosolic pH than drug-sensitive normal parental ce...
Chromosoma, 1985
By using simultaneously the AgNOR silver staining method, back-scattered electron imaging mode an... more By using simultaneously the AgNOR silver staining method, back-scattered electron imaging mode and stereo-tilt in scanning electron microscopy (SEM), it is possible to observe the nucleus through the cell surface, the nucleolus, and the tri-dimensional distribution of the Ag-NOR-associated acidic proteins. In C3H10T1:2 cells and their 7-12-dimethylbenz-alpha-anthracene-treated transformants, the staining demonstrates several intranucleolar silver-staining granules (SSG), surrounded by a weakly staining region. The SSG may represent the fibrillar center (FC) and the weakly staining region, the fibrillar dense component (FD). This component can link several SSG together to form a "rope-like structure". In cells with no visible nucleolus and inactive nucleolar organizer regions (NORs) the silver-staining granules are less numerous, close together and the presumed fibrillar dense components are not visible. The SSG are located more peripherally, and the weakly staining region and the "rope-like structure" are less prominent in control cell nucleoli than in transformed cells with a comparatively high rate of RNA synthesis.
International Journal of Cancer, 1991
We isolated an IgG2a murine monoclonal antibody (MAb) termed MAb57, specifically reactive with mu... more We isolated an IgG2a murine monoclonal antibody (MAb) termed MAb57, specifically reactive with multi-drug-resistant (MDR) human cells. Its specificity toward the MDRI gene product (P-glycoprotein) has been demonstrated by the concordant segregation of the MAb57 epitope with the MDRI gene in interspecific mouse x human cell hybrids, and the reactivity of several different MDRI gene-expressing cells with MAb57, particularly insect cells acutely infected with a baculovirus encoding the MDRI gene. MAb57 can be used to detect, by flow cytometry, variations in the relative drug-resistance levels of several MDR KB and CEM cell variants. This immunological probe has also proven useful in selectively destroying MDR target cells in an antibody-dependent cell-mediated (ADCC) assay system as well as in detecting P-glycoprotein expression in normal and malignant tissues and cells.
Stain Technology, Jun 1, 1984
Some recently developed silver staining methods allow selective staining of acidic nucleolar prot... more Some recently developed silver staining methods allow selective staining of acidic nucleolar proteins. Pretreating deparaffinized sections with Schiff's reagent improves the specificity of Goodpasture and Bloom's AgNOR staining (as modified by Kodama et al.) after aldehyde fixation.
Acta Histochemica Et Cytochemica, 1987
FEBS Letters, 1993
Multidrug-resistant KB-V1 cells carry amplified mdrl gene sequences located in an extrachromosoma... more Multidrug-resistant KB-V1 cells carry amplified mdrl gene sequences located in an extrachromosomal compartment (on episomes). Since episomes do not contain centromeric or telomeric sequences it is unclear whether they are able to bind to nuclear matrix proteins that may regulate episomal gene expression. Using high salt treatments followed by in situ hybridization and dot blot analyses we found evidence for direct binding of episomal DNA to nuclear matrix proteins. This binding could only be reversed after incubation with trypsin or proteinase K as determined by contour-clamped homogeneous electric field (CHEF) electrophoresis. Our findings are consistent with the concept that circular extrachromosomal DNA may not only reintegrate into nuclear DNA but may also be subject to functional control by regulatory proteins within the nuclear matrix.
A method for the observation of junctional complexes by backscattered electron imaging in scannin... more A method for the observation of junctional complexes by backscattered electron imaging in scanning electron microscopy, in tissue blocks, is presented. The junctional complexes are revealed by a modified silver staining method (originally devised for nucleolar organizer regions), used "en bloc" after formalin or glutaraldehyde fixation. Backscattered electron imaging allows, after this staining, the observation of junctional complexes through the surface of intact superficial cells, in the three tissues studied (liver, jejunum and urinary bladder). The interest of this approach is to offer the possibility of observing junctional complexes "by transparence," in nondissociated and nonsectioned tissues.
Proceedings of the National Academy of Sciences of the United States of America, Nov 1, 1987
Monoclonal antibody MRK16 was used to determine the location of P-glycoprotein, the product of th... more Monoclonal antibody MRK16 was used to determine the location of P-glycoprotein, the product of the multidrug-resistance gene (MDR1), in normal human tissues. The protein was found to be concentrated in a small number of specific sites. Most tissues examined revealed very little P-glycoprotein. However, certain cell types in liver, pancreas, kidney, colon, and jejunum showed specific localization of P-glycoprotein. In liver, P-glycoprotein was found exclusively on the biliary canalicular front of hepatocytes and on the apical surface of epithelial cells in small biliary ductules. In pancreas, P-glycoprotein was found on the apical surface of the epithelial cells of small ductules but not larger pancreatic ducts. In kidney, P-glycoprotein was found concentrated on the apical surface of epithelial cells of the proximal tubules. Colon and jejunum both showed high levels of P-glycoprotein on the apical surfaces of superficial columnar epithelial cells. Adrenal gland showed high levels of P-glycoprotein diffusely distributed on the surface of cells in both the cortex and medulla. These results suggest that the protein has a role in the normal secretion of metabolites and certain anti-cancer drugs into bile, urine, and directly into the lumen of the gastrointestinal tract.
Molecular and Cellular Biology of Multidrug Resistance in Tumor Cells, 1991
Cancer research, 1991
The effect of expression of the c-Ha-ras oncogene on cisplatin (DDP) sensitivity was examined in ... more The effect of expression of the c-Ha-ras oncogene on cisplatin (DDP) sensitivity was examined in murine NIH 3T3 cells transfected with the dexamethasone (DEX)-inducible mouse mammary tumor virus promoter linked to an activated c-Ha-ras gene [LTR H-ras(A) cells]. Treatment of these cells with 5 microM DEX for 24 h induced c-Ha-ras expression and produced an 8.2 +/- 1.3-fold (SD) increase in DDP resistance as quantitated by clonogenic assay. Induction of the c-Ha-ras oncogene reduced DDP accumulation by 40% and intrastrand adduct formation by 17%. In nontransfected wild-type NIH 3T3 cells, DEX did not induce DDP resistance nor did it decrease DDP accumulation. Induction of c-Ha-ras expression did not alter cellular glutathione content or the activity of glutathione-S-transferase in the LTR H-ras(A) cells. DEX increased cellular metallothionein content by 1.6-fold in NIH 3T3 cells and 3.3-fold in LTR H-ras(A) cells. We conclude that DEX-induced overexpression of a mutant c-Ha-ras gene ...
Platinum and Other Metal Coordination Compounds in Cancer Chemotherapy 2, 1996
Correlative Microscopy in Biology, 1987
Proceedings of the National Academy of Sciences, 1987
Monoclonal antibody MRK16 was used to determine the location of P-glycoprotein, the product of th... more Monoclonal antibody MRK16 was used to determine the location of P-glycoprotein, the product of the multidrug-resistance gene (MDRJ), in normal human tissues. The protein was found to be concentrated in a small number of specific sites. Most tissues examined revealed very little Pglycoprotein. However, certain cell types in liver, pancreas, kidney, colon, and jejunum showed specific localization of P-glycoprotein. In liver, P-glycoprotein was found exclusively on the biliary canalicular front of hepatocytes and on the apical surface of epithelial cells in small biliary ductules. In pancreas, P-glycoprotein was found on the apical surface of the epithelial cells of small ductules but not larger pancreatic ducts. In kidney, P-glycoprotein was found concentrated on the apical surface of epithelial cells of the proximal tubules. Colon and jejunum both showed high levels ofP-glycoprotein on the apical surfaces of superficial columnar epithelial cells. Adrenal gland showed high levels of P-glycoprotein diffusely distributed on the surface ofcells in both the cortex and medulla. These results suggest that the protein has a role in the normal secretion of metabolites and certain anti-cancer drugs into bile, urine, and directly into the lumen of the gastrointestinal tract.