Supplementary Figure Legends 1-5 from Notch Signaling Activated by Replication Stress–Induced Exp... more Supplementary Figure Legends 1-5 from Notch Signaling Activated by Replication Stress–Induced Expression of Midkine Drives Epithelial–Mesenchymal Transition and Chemoresistance in Pancreatic Cancer
Supplementary Table 1 from Notch Signaling Activated by Replication Stress–Induced Expression of ... more Supplementary Table 1 from Notch Signaling Activated by Replication Stress–Induced Expression of Midkine Drives Epithelial–Mesenchymal Transition and Chemoresistance in Pancreatic Cancer
Supplementary Figures 1-5 from Notch Signaling Activated by Replication Stress–Induced Expression... more Supplementary Figures 1-5 from Notch Signaling Activated by Replication Stress–Induced Expression of Midkine Drives Epithelial–Mesenchymal Transition and Chemoresistance in Pancreatic Cancer
Supplementary Table 2 from Notch Signaling Activated by Replication Stress–Induced Expression of ... more Supplementary Table 2 from Notch Signaling Activated by Replication Stress–Induced Expression of Midkine Drives Epithelial–Mesenchymal Transition and Chemoresistance in Pancreatic Cancer
Gemcitabine and 5- Fluorouracil (5-FU) are currently the standard of care in metastatic and adjuv... more Gemcitabine and 5- Fluorouracil (5-FU) are currently the standard of care in metastatic and adjuvant setting of pancreatic cancer (PC). However, overall survival has reached a plateau since PC inherits major potential of intrinsic and acquired resistance to chemotherapy. Molecular profile of PC is characterized by many client proteins (CP) of HSP90. Functional inhibition of HSP90 by Geldanamycin leads to simultaneous proteosomal degradation of multiple CP. Aim of the study was to evaluate the efficacy of HSP90-inhibitors in Gemcitabine and 5-FU resistant PC. Human PC cell lines (PaCa5061, PaCa5072 and PaCa5156) were isolated and brought in culture. The anti-proliferative effect of the different HSP90-inhibitors (17AAG, 17DMAG and 17AEPGA) was evaluated by the MTT-Assay. Molecular changes and apoptosis were shown by Western-blot (WB) analysis. All tested cell lines were resistant to Gemcitabine and 5-FU. 17AAG and the water soluble Geldanamycin derivates 17DMAG and 17AEPGA showed a high anti-proliferative activity in all tested cell lines with IC50 < 1 μM. A significant down regulation of EGFR, IGF-1R, AKT and MAPK was demonstrated by WB and initiation of apoptosis as an early event was proven by CASPASE-3 and PARP staining in WB. Our data demonstrate that HSP90 is a promising target in Gemcitabine and 5-FU resistant PC.
In vitro phenotype of HBV variants in patients with fulminant hepatitis B MARTINA STERNECK1-2 and... more In vitro phenotype of HBV variants in patients with fulminant hepatitis B MARTINA STERNECK1-2 and T. KALININA2 'Department of Liver Transplant ... Kidney International 1995; 48 (6): 1972-8. 3. Ogata N, Miller R, Ishak K, Purcell R. The complete nucleotide sequence of ...
Einleitung: Das Y-box bindende Protein 1 (YB-1) ist ein onkogener Transkriptionsfaktor, der in me... more Einleitung: Das Y-box bindende Protein 1 (YB-1) ist ein onkogener Transkriptionsfaktor, der in mehreren malignen Geweben, einschließlich dem Mammakarzinom und dem nicht kleinzelligen Bronchialkarzinom, hoch exprimiert wird. Obwohl dies auf eine Beteiligung von YB-1 bei der Tumorprogression schließen[for full text, please go to the a.m. URL]
e15609 Background: Pancreatic adenocarcinoma is a genetically highly complex and heterogenous tum... more e15609 Background: Pancreatic adenocarcinoma is a genetically highly complex and heterogenous tumor type with strong genetic instability which makes it resistant to therapy. Known amplifications of oncogenes such as KRAS or MYC and deletions of tumor suppresor genes such as CDKN2A and SMAD4 have demonstrated the importance of genetic alteration in this tumor type. METHODS We report the use of an Affymetrix Genome-Wide Human single nucleotide polymorphism (SNP) Array 6.0 (906,600 SNPs) to screen for gene copy number changes and allelic imbalances in 8 microdissected primary pancreatic tumors and 7 established pancreatic cancer cell lines. Gene Chip Human Genome U133 2.0 Array was used to make an RNA expression profile. Mutation analysis of KRAS and M-FISH analysis of cell lines was performed. RESULTS SNP arrays confirmed the presence of previously reported cytogenetic abnormalities in the cell lines and primary tumor probes, including MYC amplifikation at 8q24, gain of 17q12 (ERBB2/HER2), 7p12 (EGFR) and 12p12.1 (KRAS). KRAS mutation was seen in 71% of cell lines (5/7). We identified several alterations in signaling pathways such as Wnt/Notch Signaling and KRAS signaling. A sizeable subset ( 7 of 15 cases; 47%) showed an amplikon at 19q13.1-13.2 in which the serine/threonine kinase Mirk/Dyrk1B is localized, a downstream effector of oncogenic k-ras. There was also strong concordance between primary tumors and cell lines with respect to gains on 8q, 12p and 18q. Analysis of gene expression was used to localize potential target genes. M-FISH analysis showed complex karyotypes with chromosomal deletions in 9p and 18q, regions that are known to harbor tumor suppressor genes (CDKN2A, SMAD4 and TP53). CONCLUSIONS Several signaling pathways mediate tumor cell survival. Analysis of gene amplification and RNA expression profile provide molecular biological characteristics and an individual gene signature of the tumor which allow us to choose more efficient drugs to an individualized treatment. Pathways activated by KRAS such as DYRK1B may offer new therapeutic targets. Further functional characterization is needed to provide evidence for the actual role of any putative target gene. No significant financial relationships to disclose.
BackgroundPancreatic cancer is still associated with devastating prognosis. Real progress in trea... more BackgroundPancreatic cancer is still associated with devastating prognosis. Real progress in treatment options has still not been achieved. Therefore new models are urgently needed to investigate this deadly disease. As a part of this process we have established and characterized a new human pancreatic cancer cell line.MethodsThe newly established pancreatic cancer cell line PaCa 5061 was characterized for its morphology, growth rate, chromosomal analysis and mutational analysis of the K-ras, EGFR and p53 genes. Gene-amplification and RNA expression profiles were obtained using an Affymetrix microarray, and overexpression was validated by IHC analysis. Tumorigenicity and spontaneous metastasis formation of PaCa 5061 cells were analyzed in pfp-/-/rag2-/- mice. Sensitivity towards chemotherapy was analysed by MTT assay.ResultsPaCa 5061 cells grew as an adhering monolayer with a doubling time ranging from 30 to 48 hours. M-FISH analyses showed a hypertriploid complex karyotype with mul...
Metal-dependent cblA-mediated mechanism of transcription regulation of NHase could not discrimina... more Metal-dependent cblA-mediated mechanism of transcription regulation of NHase could not discriminate Ni and Co, but mechanism of NHase enzyme maturation could do this.
Research article Establishment and characterization of a new human pancreatic adenocarcinoma cell... more Research article Establishment and characterization of a new human pancreatic adenocarcinoma cell line with high metastatic potential to the lung
Metal-dependent cblA-mediated mechanism of transcription regulation of NHase could not discrimina... more Metal-dependent cblA-mediated mechanism of transcription regulation of NHase could not discriminate Ni and Co, but mechanism of NHase enzyme maturation could do this.
Rhodococcus strains are important biocatalysts used for biotechnological production of acrylamide... more Rhodococcus strains are important biocatalysts used for biotechnological production of acrylamide catalysed by a nitrile hydratase (NHase) containing cobalt. This metalloenzyme is present at high intracellular concentrations representing up to 50% of the soluble proteins in Rhodococcus rhodochrous M8 strain. Cobalt ions were formerly reported to be essential for the synthesis of the NHase subunits, encoded by nhmBA structural genes in R. rhodochrous M8. To understand the regulatory mechanisms enabling high expression of the NHase structural genes by cobalt, two reporter genes coding for an acylamidase from Rhodococcus erythropolis TA37 and a nitrilase from Alcaligenes denitrificans C-32 were fused to the nhmBA promoter. It was shown that cobalt-dependent regulation of transcription occurs independently of another regulatory genes, nhmCD, involved in substrate-dependent regulation of transcription. Cobalt ions led to an increase (up to five-fold) in transcription of reporter genes co...
Anti-apoptotic proteins are suggested to be important for the normal health of neurons and synaps... more Anti-apoptotic proteins are suggested to be important for the normal health of neurons and synapses as well as for resilience to stress. In order to determine whether stressful events may influence the expression of anti-apoptotic protein Bcl-xL in the midbrain and specifically in the midbrain serotonergic (5-HT) neurons involved in neurobehavioral responses to adverse stimuli, adult male rats were subjected to short-term or chronic forced swim stress. A short-term stress rapidly increased the midbrain bcl-xl mRNA levels and significantly elevated Bcl-xL immunoreactivity in the midbrain 5-HT cells. Stress-induced increase in glucocorticoid secretion was implicated in the observed effect. The levels of bcl-xl mRNA were decreased after stress when glucocorticoid elevation was inhibited by metyrapone (MET, 150 mg/kg), and this decrease was attenuated by glucocorticoid replacement with dexamethasone (DEX; 0.2 mg/kg). Both short-term stress and acute DEX administration, in parallel with ...
Rhodococcus strains are important biocatalysts used for biotechnological production of acrylamide... more Rhodococcus strains are important biocatalysts used for biotechnological production of acrylamide catalysed by a nitrile hydratase (NHase) containing cobalt. This metalloenzyme is present at high intracellular concentrations representing up to 50% of the soluble proteins in Rhodococcus rhodochrous M8 strain. Cobalt ions were formerly reported to be essential for the synthesis of the NHase subunits, encoded by nhmBA structural genes in R. rhodochrous M8. To understand the regulatory mechanisms enabling high expression of the NHase structural genes by cobalt, two reporter genes coding for an acylamidase from Rhodococcus erythropolis TA37 and a nitrilase from Alcaligenes denitrificans C-32 were fused to the nhmBA promoter. It was shown that cobalt-dependent regulation of transcription occurs independently of another regulatory genes, nhmCD, involved in substrate-dependent regulation of transcription. Cobalt ions led to an increase (up to five-fold) in transcription of reporter genes co...
Supplementary Figure Legends 1-5 from Notch Signaling Activated by Replication Stress–Induced Exp... more Supplementary Figure Legends 1-5 from Notch Signaling Activated by Replication Stress–Induced Expression of Midkine Drives Epithelial–Mesenchymal Transition and Chemoresistance in Pancreatic Cancer
Supplementary Table 1 from Notch Signaling Activated by Replication Stress–Induced Expression of ... more Supplementary Table 1 from Notch Signaling Activated by Replication Stress–Induced Expression of Midkine Drives Epithelial–Mesenchymal Transition and Chemoresistance in Pancreatic Cancer
Supplementary Figures 1-5 from Notch Signaling Activated by Replication Stress–Induced Expression... more Supplementary Figures 1-5 from Notch Signaling Activated by Replication Stress–Induced Expression of Midkine Drives Epithelial–Mesenchymal Transition and Chemoresistance in Pancreatic Cancer
Supplementary Table 2 from Notch Signaling Activated by Replication Stress–Induced Expression of ... more Supplementary Table 2 from Notch Signaling Activated by Replication Stress–Induced Expression of Midkine Drives Epithelial–Mesenchymal Transition and Chemoresistance in Pancreatic Cancer
Gemcitabine and 5- Fluorouracil (5-FU) are currently the standard of care in metastatic and adjuv... more Gemcitabine and 5- Fluorouracil (5-FU) are currently the standard of care in metastatic and adjuvant setting of pancreatic cancer (PC). However, overall survival has reached a plateau since PC inherits major potential of intrinsic and acquired resistance to chemotherapy. Molecular profile of PC is characterized by many client proteins (CP) of HSP90. Functional inhibition of HSP90 by Geldanamycin leads to simultaneous proteosomal degradation of multiple CP. Aim of the study was to evaluate the efficacy of HSP90-inhibitors in Gemcitabine and 5-FU resistant PC. Human PC cell lines (PaCa5061, PaCa5072 and PaCa5156) were isolated and brought in culture. The anti-proliferative effect of the different HSP90-inhibitors (17AAG, 17DMAG and 17AEPGA) was evaluated by the MTT-Assay. Molecular changes and apoptosis were shown by Western-blot (WB) analysis. All tested cell lines were resistant to Gemcitabine and 5-FU. 17AAG and the water soluble Geldanamycin derivates 17DMAG and 17AEPGA showed a high anti-proliferative activity in all tested cell lines with IC50 < 1 μM. A significant down regulation of EGFR, IGF-1R, AKT and MAPK was demonstrated by WB and initiation of apoptosis as an early event was proven by CASPASE-3 and PARP staining in WB. Our data demonstrate that HSP90 is a promising target in Gemcitabine and 5-FU resistant PC.
In vitro phenotype of HBV variants in patients with fulminant hepatitis B MARTINA STERNECK1-2 and... more In vitro phenotype of HBV variants in patients with fulminant hepatitis B MARTINA STERNECK1-2 and T. KALININA2 'Department of Liver Transplant ... Kidney International 1995; 48 (6): 1972-8. 3. Ogata N, Miller R, Ishak K, Purcell R. The complete nucleotide sequence of ...
Einleitung: Das Y-box bindende Protein 1 (YB-1) ist ein onkogener Transkriptionsfaktor, der in me... more Einleitung: Das Y-box bindende Protein 1 (YB-1) ist ein onkogener Transkriptionsfaktor, der in mehreren malignen Geweben, einschließlich dem Mammakarzinom und dem nicht kleinzelligen Bronchialkarzinom, hoch exprimiert wird. Obwohl dies auf eine Beteiligung von YB-1 bei der Tumorprogression schließen[for full text, please go to the a.m. URL]
e15609 Background: Pancreatic adenocarcinoma is a genetically highly complex and heterogenous tum... more e15609 Background: Pancreatic adenocarcinoma is a genetically highly complex and heterogenous tumor type with strong genetic instability which makes it resistant to therapy. Known amplifications of oncogenes such as KRAS or MYC and deletions of tumor suppresor genes such as CDKN2A and SMAD4 have demonstrated the importance of genetic alteration in this tumor type. METHODS We report the use of an Affymetrix Genome-Wide Human single nucleotide polymorphism (SNP) Array 6.0 (906,600 SNPs) to screen for gene copy number changes and allelic imbalances in 8 microdissected primary pancreatic tumors and 7 established pancreatic cancer cell lines. Gene Chip Human Genome U133 2.0 Array was used to make an RNA expression profile. Mutation analysis of KRAS and M-FISH analysis of cell lines was performed. RESULTS SNP arrays confirmed the presence of previously reported cytogenetic abnormalities in the cell lines and primary tumor probes, including MYC amplifikation at 8q24, gain of 17q12 (ERBB2/HER2), 7p12 (EGFR) and 12p12.1 (KRAS). KRAS mutation was seen in 71% of cell lines (5/7). We identified several alterations in signaling pathways such as Wnt/Notch Signaling and KRAS signaling. A sizeable subset ( 7 of 15 cases; 47%) showed an amplikon at 19q13.1-13.2 in which the serine/threonine kinase Mirk/Dyrk1B is localized, a downstream effector of oncogenic k-ras. There was also strong concordance between primary tumors and cell lines with respect to gains on 8q, 12p and 18q. Analysis of gene expression was used to localize potential target genes. M-FISH analysis showed complex karyotypes with chromosomal deletions in 9p and 18q, regions that are known to harbor tumor suppressor genes (CDKN2A, SMAD4 and TP53). CONCLUSIONS Several signaling pathways mediate tumor cell survival. Analysis of gene amplification and RNA expression profile provide molecular biological characteristics and an individual gene signature of the tumor which allow us to choose more efficient drugs to an individualized treatment. Pathways activated by KRAS such as DYRK1B may offer new therapeutic targets. Further functional characterization is needed to provide evidence for the actual role of any putative target gene. No significant financial relationships to disclose.
BackgroundPancreatic cancer is still associated with devastating prognosis. Real progress in trea... more BackgroundPancreatic cancer is still associated with devastating prognosis. Real progress in treatment options has still not been achieved. Therefore new models are urgently needed to investigate this deadly disease. As a part of this process we have established and characterized a new human pancreatic cancer cell line.MethodsThe newly established pancreatic cancer cell line PaCa 5061 was characterized for its morphology, growth rate, chromosomal analysis and mutational analysis of the K-ras, EGFR and p53 genes. Gene-amplification and RNA expression profiles were obtained using an Affymetrix microarray, and overexpression was validated by IHC analysis. Tumorigenicity and spontaneous metastasis formation of PaCa 5061 cells were analyzed in pfp-/-/rag2-/- mice. Sensitivity towards chemotherapy was analysed by MTT assay.ResultsPaCa 5061 cells grew as an adhering monolayer with a doubling time ranging from 30 to 48 hours. M-FISH analyses showed a hypertriploid complex karyotype with mul...
Metal-dependent cblA-mediated mechanism of transcription regulation of NHase could not discrimina... more Metal-dependent cblA-mediated mechanism of transcription regulation of NHase could not discriminate Ni and Co, but mechanism of NHase enzyme maturation could do this.
Research article Establishment and characterization of a new human pancreatic adenocarcinoma cell... more Research article Establishment and characterization of a new human pancreatic adenocarcinoma cell line with high metastatic potential to the lung
Metal-dependent cblA-mediated mechanism of transcription regulation of NHase could not discrimina... more Metal-dependent cblA-mediated mechanism of transcription regulation of NHase could not discriminate Ni and Co, but mechanism of NHase enzyme maturation could do this.
Rhodococcus strains are important biocatalysts used for biotechnological production of acrylamide... more Rhodococcus strains are important biocatalysts used for biotechnological production of acrylamide catalysed by a nitrile hydratase (NHase) containing cobalt. This metalloenzyme is present at high intracellular concentrations representing up to 50% of the soluble proteins in Rhodococcus rhodochrous M8 strain. Cobalt ions were formerly reported to be essential for the synthesis of the NHase subunits, encoded by nhmBA structural genes in R. rhodochrous M8. To understand the regulatory mechanisms enabling high expression of the NHase structural genes by cobalt, two reporter genes coding for an acylamidase from Rhodococcus erythropolis TA37 and a nitrilase from Alcaligenes denitrificans C-32 were fused to the nhmBA promoter. It was shown that cobalt-dependent regulation of transcription occurs independently of another regulatory genes, nhmCD, involved in substrate-dependent regulation of transcription. Cobalt ions led to an increase (up to five-fold) in transcription of reporter genes co...
Anti-apoptotic proteins are suggested to be important for the normal health of neurons and synaps... more Anti-apoptotic proteins are suggested to be important for the normal health of neurons and synapses as well as for resilience to stress. In order to determine whether stressful events may influence the expression of anti-apoptotic protein Bcl-xL in the midbrain and specifically in the midbrain serotonergic (5-HT) neurons involved in neurobehavioral responses to adverse stimuli, adult male rats were subjected to short-term or chronic forced swim stress. A short-term stress rapidly increased the midbrain bcl-xl mRNA levels and significantly elevated Bcl-xL immunoreactivity in the midbrain 5-HT cells. Stress-induced increase in glucocorticoid secretion was implicated in the observed effect. The levels of bcl-xl mRNA were decreased after stress when glucocorticoid elevation was inhibited by metyrapone (MET, 150 mg/kg), and this decrease was attenuated by glucocorticoid replacement with dexamethasone (DEX; 0.2 mg/kg). Both short-term stress and acute DEX administration, in parallel with ...
Rhodococcus strains are important biocatalysts used for biotechnological production of acrylamide... more Rhodococcus strains are important biocatalysts used for biotechnological production of acrylamide catalysed by a nitrile hydratase (NHase) containing cobalt. This metalloenzyme is present at high intracellular concentrations representing up to 50% of the soluble proteins in Rhodococcus rhodochrous M8 strain. Cobalt ions were formerly reported to be essential for the synthesis of the NHase subunits, encoded by nhmBA structural genes in R. rhodochrous M8. To understand the regulatory mechanisms enabling high expression of the NHase structural genes by cobalt, two reporter genes coding for an acylamidase from Rhodococcus erythropolis TA37 and a nitrilase from Alcaligenes denitrificans C-32 were fused to the nhmBA promoter. It was shown that cobalt-dependent regulation of transcription occurs independently of another regulatory genes, nhmCD, involved in substrate-dependent regulation of transcription. Cobalt ions led to an increase (up to five-fold) in transcription of reporter genes co...
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