Generation of a Cell Line Selectively Producing Functionally Active OATP1B1 Transporter.

The solute carrier organic anion transporter family member, OATP1B1, is one of the most important transporter proteins, which mediate penetration of many endogenous substances and xenobiotics into hepatocytes. A model system providing expression of the functional protein is needed to assess interaction of OATP1B1 with various substances. Based on the HEK293 cells, we obtained the HEK293-OATP1B1 cell line, constitutively expressing the SLCO1B1 gene encoding the OATP1B1 transporter. Expression of the SLCO1B1 gene was confirmed by real-time PCR analysis and Western blotting. Functionality of the transporter was assessed by the transport of atorvastatin, which is a substrate of OATP1B1. Cells of the resulting cell line, which selectively express the functionally active recombinant OATP1B1 transporter, can be used to study functions of the protein and to test drugs for being substrates, inducers, and inhibitors of OATP1B1, and to assess the risks of drug interactions.

Yeast Rpn4 Links the Proteasome and DNA Repair via RAD52 Regulation.

Environmental and intracellular factors often damage DNA, but multiple DNA repair pathways maintain genome integrity. In yeast, the 26S proteasome and its transcriptional regulator and substrate Rpn4 are involved in DNA damage resistance. Paradoxically, while proteasome dysfunction may induce hyper-resistance to DNA-damaging agents, Rpn4 malfunction sensitizes yeasts to these agents. Previously, we proposed that proteasome inhibition causes Rpn4 stabilization followed by the upregulation of Rpn4-dependent DNA repair genes and pathways. Here, we aimed to elucidate the key Rpn4 targets responsible for DNA damage hyper-resistance in proteasome mutants. We impaired the Rpn4-mediated regulation of candidate genes using the CRISPR/Cas9 system and tested the sensitivity of mutant strains to 4-NQO, MMS and zeocin. We found that the separate or simultaneous deregulation of 19S or 20S proteasome subcomplexes induced MAG1, DDI1, RAD23 and RAD52 in an Rpn4-dependent manner. Deregulation of RAD23, DDI1 and RAD52 sensitized yeast to DNA damage. Genetic, epigenetic or dihydrocoumarin-mediated RAD52 repression restored the sensitivity of the proteasome mutants to DNA damage. Our results suggest that the Rpn4-mediated overexpression of DNA repair genes, especially RAD52, defines the DNA damage hyper-resistant phenotype of proteasome mutants. The developed yeast model is useful for characterizing drugs that reverse the DNA damage hyper-resistance phenotypes of cancers.

Deregulation of the 19S proteasome complex increases yeast resistance to 4-NQO and oxidative stress via upregulation of Rpn4- and proteasome-dependent stress responsive genes.

The 26S proteasome participates in cell stress responses via its ability to degrade regulatory and damaged proteins. In yeast, mutations in the subunits of the 19S proteasome regulatory subcomplex cause hyper-resistance to 4-nitroquinoline-1-oxide (4-NQO), a chemical mutagen and carcinogen. These data suggest a negative role for the 19S proteasome complex in the cellular response to 4-NQO, although the underlying mechanism is not clear. We proposed that decreased 19S subcomplex activity leads to the stabilisation of Rpn4p, a transcription factor and proteasome substrate. In turn, stabilised Rpn4p may upregulate stress-responsive genes that participate in the response to 4-NQO-induced stress. To test our hypothesis, we impaired the expression of the RPT5 gene, which encodes the ATPase subunit of the 19S subcomplex, by mutating the Rpn4p binding site in its promoter. The mutant strain accumulates polyubiquitinated proteins-a hallmark of compromised proteasome function-and shows hyper-resistance to 4-NQO. We found several groups of genes that conferred resistance to 4-NQO-induced stress and were overexpressed due to the Rpn4p stabilisation and impaired 19S subcomplex function. The upregulated genes are involved in the oxidative and proteotoxic stress response pathways, multidrug resistance and biosynthesis of cysteine and methionine. Consistently, the mutant strain was hyper-resistant to oxidative stress. Our data imply that the ubiquitin-proteasome system may regulate the cellular response to 4-NQO at the transcriptional level.