Holger Woehlecke

Reversal of breast cancer resistance protein-mediated drug resistance by tryprostatin A.

MDR in human cancers is one of the major causes of failure of chemotherapy. A member of the superfamily of ABC transporters, BCRP, was demonstrated to confer an atypical MDR phenotype to tumor cells. To overcome the BCRP‐mediated drug resistance, the fungal secondary metabolite TPS‐A, a diketopiperazine, was analyzed with regard to its potency to reverse the BCRP‐mediated drug‐resistant phenotype. At concentrations of 10–50 μM, TPS‐A reversed a mitoxantrone‐resistant phenotype and inhibited the cellular BCRP‐dependent mitoxantrone accumulation in the human gastric carcinoma cell line EPG85‐257RNOV, the human breast cancer cell line MCF7/AdrVp (both exhibiting acquired BCRP‐mediated MDR) and the BCRP cDNA‐transfected breast cancer cell line MCF‐7/BCRP clone 8. No cytotoxicity was seen at effective concentrations. These data indicate that TPS‐A is a novel BCRP inhibitor.

Enhanced exposure of phosphatidylserine in human gastric carcinoma cells overexpressing the half-size ABC transporter BCRP (ABCG2)

Members of the ABC (ATP-binding cassette) transporter super-family are emerging to be involved in lipid transport. In the present study, we studied the organization of phospholipids in the plasma membrane of EPG85-257 human gastric carcinoma cells overexpressing BCRP (breast cancer resistance protein, ABCG-2), a half-size transporter belonging to the ABCG subfamily. A significantly increased plasma membrane association of the PS (phosphatidylserine)-binding probe FITC-Annexin V in comparison with control cells was observed. Treatment of BCRP -overexpressing cells with the inhibitor Tryprostatin A decreased FITC-Annexin V binding almost to the control level. This suggests an enhanced exposure of PS in BCRP -overexpressing cells, which is dependent on functional BCRP. A role of BCRP in the transverse distribution of lipids in the plasma membrane is supported by the increased outward transport of the lipid analogue C6- N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-PS in BCRP -overexpressing EPG85-257 cells and MCF-7 human breast cancer cells. As shown for BCRP -overexpressing EPG85-257 cells, enhanced outward redistribution of the lipid analogue is inhibited by Tryprostatin A as well as by reduction of BCRP expression on transfection with an anti- BCRP -ribozyme. We also observed an enhanced outward transport of C6- N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phosphatidylcholine in BCRP -overexpressing EPG85-257 cells, suggesting that the influence of BCRP on transverse lipid organization is not highly specific.

Cell wall microcapsules with different porosity from suspension cultured Chenopodium album

Abstract The primary cell wall of suspension cultured higher plant tissues may be used for size fractionation of macromolecules of different ranges of molecular mass by permeation chromatography, as the size limits of cell wall penetration may be adjusted by controlled decay of polygalacturonan. If care is taken to prevent pectin decay by β-elimination during the preparation of the vesicular packing material (VP) from suspension grown cell clusters of Chenopodium album (low temperature, pectin-preserving proteolysis buffer and washing solutions), extraction with ethanol and deproteinization with a pancreatic enzyme mixture did not significantly increase the size limit of permeation (SLP) of the native cell wall. However, the SLP was significantly increased after long term washing at room temperature or heat treatment with phosphate buffer at pH 7. If the VP was incubated in 2% Na 2 CO 3 solution at room temperature, its SLP increased significantly within the first 30 min but remained stable on further incubation in the alkaline solution. The extent of the porosity change of the cell wall occurring in Na 2 CO 3 solution may be varied by choice of the incubation temperature. Permeation chromatography using the variants of the VP with increased porosity enables separation between proteins with large peak separation in the range of middle (50–100 kD) and large (300 kD) molecular mass.

Extraction of nucleic acids from yeast cells and plant tissues using ethanol as medium for sample preservation and cell disruption

Here we report that dehydrated ethanol is an excellent medium for both in situ preservation of nucleic acids and cell disruption of plant and yeast cells. Cell disruption was strongly facilitated by prior dehydration of the ethanol using dehydrated zeolite. Following removal of ethanol, nucleic acids were extracted from the homogenate pellet using denaturing buffers. The method provided DNA and RNA of high yield and integrity. Whereas cell wall disruption was essential for extraction of DNA and large RNA molecules, smaller molecules such as tRNAs could be selectively extracted from undisrupted, ethanol-treated yeast cells. Our results demonstrate the utility of absolute ethanol for sample fixation, cell membrane and cell wall disruption, as well as preservation of nucleic acids during sample storage.