Stressing the Ubiquitin-Proteasome System without 20S Proteolytic Inhibition Selectively Kills Cervical Cancer Cells

Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate, and for specific signaling pathways, notably HPV E6-targeted degradation of p53 and PDZ proteins. Natural compounds with antioxidant properties including flavonoids and triterpenoids hold promise as anticancer agents by interfering with ubiquitin-dependent protein degradation. An increasing body of evidence indicates that their α-β unsaturated carbonyl system is the molecular determinant for inhibition of ubiquitin-mediated protein degradation up-stream of the catalytic sites of the 20S proteasome. Herein we report the identification and characterization of a new class of chalcone-based, potent and cell permeable chemical inhibitors of ubiquitin-dependent protein degradation, and a lead compound RAMB1. RAMB1 inhibits ubiquitin-dependent protein degradation without compromising the catalytic activities of the 20S proteasome, a mechanism distinct from that of Bortezomib. Treatment of cervical cancer cells with RAMB1 triggers unfolded protein responses, including aggresome formation and Hsp90 stabilization, and increases p53 steady state levels. RAMB1 treatment results in activation of lysosomal-dependent degradation pathways as a mechanism to compensate for increasing levels of poly-ubiquitin enriched toxic aggregates. Importantly, RAMB1 synergistically triggers cell death of cervical cancer cells when combined with the lysosome inhibitor Chloroquine

Binding of hnRNP L to the Pre-mRNA Processing Enhancer of the Herpes Simplex Virus Thymidine Kinase Gene Enhances both Polyadenylation and Nucleocytoplasmic Export of Intronless mRNAs

Liu and Mertz (Genes Dev. 9:1766-1780, 1995) previously identified a 119-nt pre-mRNA processing enhancer (PPE) element within the herpes simplex virus type 1 thymidine kinase gene that enables intron-independent gene expression in higher eukaryotes by binding heterogeneous nuclear ribonucleoprotein L (hnRNP L). Here, we identify a 49-nt subelement within this PPE that enhanced stability, polyadenylation, and cytoplasmic accumulation of transcripts synthesized in CV-1 cells from an intronless variant of the human β-globin gene when present in two or more tandem copies. This 2×TK49 PPE also enhanced (i) the efficiency of polyadenylation of intronless β-globin RNA in a cell-free polyadenylation system and (ii) the kinetics of nucleocytoplasmic export of an intronless variant of adenovirus major late leader region RNA in Xenopus oocytes. This 2×TK49 PPE bound only hnRNP L. Analysis of 2×TK49 PPE mutants showed a strong positive correlation existed between binding hnRNP L and enhancement of intronless β-globin gene expression. hnRNP L was found to associate with both the mRNA export factor TAP and the exon-exon junction complex protein Aly/REF. Thus, we conclude that hnRNP L plays roles in enhancing stability, polyadenylation, and nucleocytoplasmic export; it does so, at least in part, by directly recruiting to intronless PPE-containing RNAs cofactors normally recruited to intron-containing RNAs