ABSTRACT
PRMT7 belongs to the protein arginine methyl-transferases family. We show that downregulation of PRMT7alpha and beta isoforms in DC-3F hamster cells was associated with increased sensitivity to the Top1 inhibitor camptothecin (CPT). This effect was not due to a change in Top1 contents or catalytic activity, or to a difference in the reversal of DNA breaks. Overexpression of PRMT7alpha and beta in DC-3F cells had no effect on CPT sensitivity, whereas it conferred a resistance to DC-3F/9-OH-E cells for which both isoforms are reduced by two- to three-fold as compared to DC-3F parental cells. Finally, downregulation of the human PRMT7 could also sensitize HeLa cells to CPT, suggesting that it could be used as a target to potentiate CPT derivatives.
Subject(s)
Antineoplastic Agents/pharmacology , Camptothecin/pharmacology , Drug Resistance, Neoplasm/genetics , Methyltransferases/genetics , Neoplasms/enzymology , Animals , Cell Line, Tumor , Cricetinae , Down-Regulation , HeLa Cells , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Methyltransferases/metabolism , Oligonucleotides, Antisense/genetics , Protein-Arginine N-MethyltransferasesABSTRACT
Unr (upstream of N-ras) is a cytoplasmic RNA-binding protein that can act as a regulator of mRNA stability and IRES-mediated translation. Unr, a member of the cold-shock domain (CSD) protein super-family, is ubiquitously expressed, with variable abundance, in different tissues or during embryonic development. Prokaryotic and eukaryotic cold-shock protein expression is highly regulated at both the transcriptional and post-transcriptional levels. Here we analyzed the role of the 5'- and 3'-untranslated regions (UTR) of unr mRNA in post-transcriptional regulation of Unr expression. We show that, in vitro, unr 3'-UTR specifically destabilizes unr transcripts. Accordingly, in vivo, the half-life of unr messages deleted of noncoding regions is increased by approximately 3.6 fold, resulting in an enhanced steady-state level of Unr protein. We also show that the 5'-UTR exhibits IRES activity both when translated in vitro and in transiently transfected cells. This IRES activity displays cell type specificity with a higher efficiency in HeLa and HuH7 than in ES cells. Moreover, Unr IRES activity was higher in unr(-/-) than in unr(+/+) ES cells, indicating that Unr negatively regulates its own IRES activity. Our studies further reveal that Unr specifically interacts with its own mRNAs in vivo. These results suggest that a feedback control of mRNA translation is involved in regulating Unr expression.
Subject(s)
3' Untranslated Regions/physiology , 5' Untranslated Regions/physiology , Gene Expression Regulation/physiology , Poly(A)-Binding Proteins/genetics , RNA Stability/physiology , RNA, Messenger/physiology , Amino Acid Sequence , Animals , Base Sequence , Binding Sites/physiology , Cell Line , Mice , Molecular Sequence Data , Poly(A)-Binding Proteins/biosynthesisABSTRACT
A comparison was made between two K vitamin analogs. Growth in vitro of Hep G2 hepatoma cells was inhibited both by Compound 5 (Cpd 5), a recently synthesized thioalkyl analog of vitamin K or 2-(2-mercaptoethanol)-3-methyl-1, 4-naphthoquinone, as well as by synthetic vitamin K3 (menadione). Using synchronized Hep G2 hepatoma cells, the actions of both Cpd 5 and vitamin K3 on cell cycle regulating proteins were examined. Cpd 5 decreased the levels of cyclin D1, Cdk4, p16, p21 and cyclin B1. By contrast, VK3 only decreased the level of cyclin D1, but had no effect on the levels of Cdk4, p16 or p21. Interestingly, both VK3 and VK2 increased the levels of p21. The naturally occurring K vitamins had little effect on cell growth and none on the cyclins or Cdks. Amounts and activity of the G1/S phase controlling Cdc25A were measured. We found that Cpd 5 directly inhibited both Cdc25A activity and its protein expression, whereas VK3 did not. Thus, the main effects of Cpd 5 were on G1 and S phase proteins, especially Cdk4 and Cdc25A amounts in contrast to VK3. Computer docking studies of Cpd 5 and VK3 to Cdc25A phosphatase showed three binding sites. In the best conformation, Cpd 5 was found to be closer to the enzyme active site than VK3. These findings show that Cpd 5 represents a new class of anticancer agent, being a protein tyrosine phosphatase (PTP) antagonist, that binds to Cdc25A with suppression of its activity. Tumors expressing high levels of oncogenic Cdc25A phosphatase may thus be susceptible to the growth inhibitory activities of this class of compound.
