Phospholipase A2 inhibition at different substrate concentrations.
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Biomedical subjects
Markus Böhl
Publications and source records attributed to Markus Böhl.
A cellular model system for expression studies of coagulation proteins.
INTRODUCTION: The development of novel antithrombotic agents directly affecting gene expression requires well established, reliable and useful in vitro model systems for initial validation of drug effects. Since most proteins involved in coagulation are synthesized by the liver, the hepatoblastoma cell line Hep G2 is introduced, here, as a model system to test nucleic acid based coagulation inhibitors. METHODS: Hep G2 cells were characterized with respect to prothrombin, tissue factor and factor VIII expression in dependence of cell culture conditions. Reliable enzyme linked immuno sorbent assays as well as viability tests were introduced that allow drug screening procedures with multiple probes in microplate format. Furthermore, a multiplex PCR-procedure has been presented that offers the possibility to simultaneously detect the effects of a selected compound on two coagulation proteins in comparison to a house keeping gene. RESULTS: Hep G2 cells were not affected in viability by cell culture conditions, while proliferation and the expression patterns of some coagulation factors were affected by the adhesion factor collagen. The prothrombin expression characteristics allowed us to choose a specific time point for the transfection of Hep G2 cells with prothrombin specific antisense oligonucleotides. Antisense oligonucleotides inhibited prothrombin expression independent from culture conditions and the effects were detected on protein-and mRNA-level. DISCUSSION: Nucleic acid based agents require cellular in vitro model systems since they affect the process of gene expression and not the gene product. Hep G2 cells are a useful model to study effects of novel nucleic acid based coagulation inhibitors with an antisense mechanism of action on protein and mRNA level.
Blood Coagulation Factors↗
A potent inhibitor of prothrombin gene expression as a result of standardized target site selection and design of antisense oligonucleotides.
The development of antisense oligonucleotides (AS-ODN) always had the limitation that because of complex mRNA secondary structures, not every designed AS-ODN inhibited the expression of its target. There have been many investigations to overcome this problem in the last few years. This produced a great deal of theoretical and empirical findings about characteristics of effective AS-ODNs in respect to their target regions but no standardized selection procedure of AS-ODN target regions within a given mRNA or standardized design of AS-ODNs against a specific target region. We present here a standardized method based on secondary structure prediction for target site selection and AS-ODN design, followed by validation of the antisense effect caused by our predicted AS-ODNs in cell culture. The combination of theoretical design and experimental selection procedure led to an AS-ODN that efficiently and specifically reduces prothrombin mRNA and antigen.
Cell Line, Tumor↗