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1.
Pancreas ; 40(1): 144-50, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20938367

ABSTRACT

OBJECTIVES: RNA interference, an indispensable tool in functional genomics, can be induced by small interfering RNAs (siRNAs). Because of the transient nature of siRNA-mediated RNA interference, the continuous use of transfection reagents is mandatory. Because transfection reagents are expensive, cost-effective alternatives must be considered. In this study, we describe a polyethylenimine-based siRNA transfection protocol for pancreatic cancer cell lines. METHODS: For determination of polyethylenimine-based transfection efficiency, a FAM-labeled siRNA was transfected into several pancreatic cancer cell lines and subsequently analyzed by flow cytometry. The effective knockdown of 2 siRNAs was determined on the protein level by Western blot. Toxicity of the transfection reagent was analyzed by viability assays. RESULTS: Polyethylenimine can be used without overt cellular morphological changes, and toxicity is negligible in human and murine pancreatic cancer cell lines. Transfection efficiencies ranged between 83% and 98% in the cell lines used. The knockdown at the protein level was comparable to commercially available transfection reagents. Polyethylenimine and siRNA concentrations, incubation time, and cell density are determinates of the transfection efficiency. CONCLUSIONS: Polyethylenimine is a suitable and cost-effective alternative for transfecting siRNAs into pancreatic cancer cells.


Subject(s)
Pancreatic Neoplasms/genetics , Polyethyleneimine/pharmacology , RNA, Small Interfering/genetics , Transfection/methods , Animals , Cell Count , Cell Line, Tumor , Cell Survival/drug effects , Cost-Benefit Analysis , Humans , Mice , NF-kappa B/metabolism , Phenotype
2.
Mol Cancer ; 9: 80, 2010 Apr 16.
Article in English | MEDLINE | ID: mdl-20398369

ABSTRACT

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors with a dismal prognosis and no effective conservative therapeutic strategies. Although it is demonstrated that histone deacetylases (HDACs), especially the class I HDACs HDAC1, 2 and 3 are highly expressed in this disease, little is known about HDAC isoenzyme specific functions. RESULTS: Depletion of HDAC2, but not HDAC1, in the pancreatic cancer cell lines MiaPaCa2 and Panc1 resulted in a marked sensitization towards the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Correspondingly, the more class I selective HDAC inhibitor (HDACI) valproic acid (VPA) synergized with TRAIL to induce apoptosis of MiaPaCa2 and Panc1 cells. At the molecular level, an increased expression of the TRAIL receptor 1 (DR5), accelerated processing of caspase 8, pronounced cleavage of the BH3-only protein Bid, and increased effector caspase activation was observed in HDAC2-depleted and TRAIL-treated MiaPaCa2 cells. CONCLUSIONS: Our data characterize a novel HDAC2 function in PDAC cells and point to a strategy to overcome TRAIL resistance of PDAC cells, a prerequisite to succeed with a TRAIL targeted therapy in clinical settings.


Subject(s)
Apoptosis/physiology , Carcinoma, Pancreatic Ductal/metabolism , Histone Deacetylase 2/metabolism , Pancreatic Neoplasms/metabolism , TNF-Related Apoptosis-Inducing Ligand/metabolism , Apoptosis/drug effects , Blotting, Western , Carcinoma, Pancreatic Ductal/genetics , Cell Line, Tumor , Enzyme Inhibitors/pharmacology , Gene Expression , Gene Expression Regulation, Neoplastic , Histone Deacetylase 1/genetics , Histone Deacetylase 1/metabolism , Histone Deacetylase 2/genetics , Humans , Pancreatic Neoplasms/genetics , RNA, Small Interfering , Receptors, TNF-Related Apoptosis-Inducing Ligand/biosynthesis , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Valproic Acid/pharmacology
3.
J Cell Mol Med ; 14(6A): 1255-63, 2010 Jun.
Article in English | MEDLINE | ID: mdl-19929947

ABSTRACT

Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with a median survival below 6 months and a 5-year survival rate below 1%. Effective therapies for locally advanced or metastatic tumours are missing and curatively resected patients relapse in over 80% of the cases. Although histone deacetylases (HDACs) are involved in the control of proliferation, apoptosis, differentiation, migration and angiogenesis of cancer cells, knowledge about the expression patterns and functions of individual HDAC isoenzymes in pancreatic cancer is sparse. This review summarizes the roles of HDACs as novel therapeutic targets and the molecular mode of action of HDAC-inhibitors (HDACI) in PDACs. Success of HDACI in clinical settings will depend on an increased knowledge of HDAC functions as well as on a better understanding of the mode of action of HDACI. Pre-clinical experimental data that constitute the basis for rational therapeutic strategies to treat PDAC are described here. Translating these rational-based therapies into the clinic will finally increase our chance to establish an effective HDACI-containing combination therapy effective against PDAC.


Subject(s)
Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/enzymology , Histone Deacetylase Inhibitors/therapeutic use , Histone Deacetylases/metabolism , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/enzymology , Apoptosis/drug effects , Carcinoma, Pancreatic Ductal/pathology , Cell Proliferation/drug effects , Histone Deacetylase Inhibitors/pharmacology , Humans , Neoplasm Metastasis
4.
Biol Chem ; 385(10): 975-85, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15551873

ABSTRACT

We have mutated the monomer-monomer interface of the restriction endonuclease EcoRI in order to destabilize the homodimer and to stabilize heterodimers. Mutations of Leu158 to charged amino acid residues result in strong destabilization of the dimer. The largest effect was detected for the L158D mutant which is monomeric even at higher concentrations. It unspecifically degrades DNA by cleaving both single strands independently every 15 nucleotides on the average. Although cleavage is reproducible, it is not determined by nucleotide sequence but by general properties like conformation or deformability as has been found for other unspecific nucleases. Mutations of Ile230, which is in direct contact with Leu158 of the other subunit, cause structural changes with the loss of about ten percent alpha-helix content, but interfere only marginally with homodimerization and double strand cleavage. Again the mutation to aspartate shows the strongest effects. Mixtures of single mutants, one containing aspartate at one of the two positions and the other lysine at the corresponding position, form heterodimers. These are mainly stabilized compared to the homodimers by re-establishment of the wild-type hydrophobic interaction at the not mutated residues while an interaction of aspartate and lysine seems energetically unfavorable in this structural context.


Subject(s)
DNA, Bacterial/genetics , Deoxyribonuclease EcoRI/genetics , Mutation , Sequence Analysis, DNA/methods , DNA Mutational Analysis/methods , DNA Restriction Enzymes/chemistry , DNA Restriction Enzymes/genetics , DNA, Bacterial/chemistry , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Deoxyribonuclease EcoRI/chemistry , Protein Conformation
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