Serum depletion of holo-ceruloplasmin induced by silver ions in vivo reduces uptake of cisplatin.

There is an emerging link between extracellular copper concentration and the uptake of cisplatin mediated by copper transporter CTR1 in cell cultures and unicellular eukaryotes. To test the link between extracellular copper level and cisplatin uptake by organs in vivo we used mice with low copper status parameters induced by AgCl-containing diet (Ag-mice). In Ag-mice, serum copper status and liver copper metabolism were characterized. It was shown that the expression level of copper transporter genes and activity of ubiquitous intracellular cuproenzymes were not affected but the level of serum holo-ceruloplasmin was not detectable. Silver was selectively absorbed by liver and accumulated in the mitochondrial matrix. Silver was present in an exchangeable form and was excreted through bile. Ag-mice model is characterized by high reproducibility, reversibility, synchronicity, and definiteness of ceruloplasmin-associated copper deficiency. After cisplatin treatment Ag-mice, as compared to control mice, demonstrated the delay in platinum uptake by organs during first 30 min. This effect was not observed at later time points probably due to cisplatin induced copper release to blood, which resulted in the recovery of copper status. These data allowed us to conclude that cisplatin uptake was coupled to copper transport in vivo.

Mass spectrometry and biochemical analysis of RNA polymerase II: targeting by protein phosphatase-1.

Transcription of eukaryotic genes is regulated by phosphorylation of serine residues of heptapeptide repeats of the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII). We previously reported that protein phosphatase-1 (PP1) dephosphorylates RNAPII CTD in vitro and inhibition of nuclear PP1-blocked viral transcription. In this article, we analyzed the targeting of RNAPII by PP1 using biochemical and mass spectrometry analysis of RNAPII-associated regulatory subunits of PP1. Immunoblotting showed that PP1 co-elutes with RNAPII. Mass spectrometry approach showed the presence of U2 snRNP. Co-immunoprecipitation analysis points to NIPP1 and PNUTS as candidate regulatory subunits. Because NIPP1 was previously shown to target PP1 to U2 snRNP, we analyzed the effect of NIPP1 on RNAPII phosphorylation in cultured cells. Expression of mutant NIPP1 promoted RNAPII phosphorylation suggesting that the deregulation of cellular NIPP1/PP1 holoenzyme affects RNAPII phosphorylation and pointing to NIPP1 as a potential regulatory factor in RNAPII-mediated transcription.

e2f1 Gene is a new member of Wnt/beta-catenin/Tcf-regulated genes.

HDAC inhibitors induce cell cycle arrest of E1A+Ras-transformed cells accompanied by e2f1 gene down-regulation and activation of Wnt pathway. Here we show that e2f1 expression is regulated through the Wnt/Tcf-pathway: e2f1 promoter activity is inhibited by sodium butyrate (NaB) and by overexpression of beta-catenin/Tcf. The e2f1 promoter was found to contain two putative Tcf-binding elements: the proximal one competes well with canonical Tcf element in DNA-binding assay. Being inserted into luciferase reporter vector, the identified element provides positive transcriptional regulation in response to beta-catenin/Tcf co-transfection and NaB treatment. Thus we have firstly demonstrated that e2f1 belongs to genes regulated through Wnt/beta-catenin/Tcf pathway.