Tracking the expression of RNA in a cell-specific manner is a major challenge in basic and disease research. Herein we outline the current state of employing chemical approaches for cell-specific RNA expression studies. We define the utility of metabolic labels for tracking RNA synthesis, the approaches for characterizing metabolic incorporation and enrichment of labeled RNAs, and finally outline how these approaches have been used to study biological systems by providing mechanistic insights into transcriptional dynamics. Further efforts on this front will be the continued development of novel chemical handles for RNA enrichment and profiling as well as innovative approaches to control cell-specific incorporation of chemically modified metabolic probes. These advancements in RNA metabolic labeling techniques permit sensitive detection of RNA expression dynamics within relatively small subsets of cells in living tissues and organisms that are critical to performing complex developmental and pathological processes. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA Evolution and Genomics > Ribonomics RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry
A BS TRACT: Background: Molecules related to glucocerebrosidase (GCase) are potential biomarkers for development of compounds targeting GBA1-associated Parkinson's disease (GBA-PD). Objectives: Assessing variability of various glycosphingolipids (GSLs) in plasma, peripheral blood mononuclear cells (PBMCs), and cerebrospinal fluid (CSF) across GBA-PD, idiopathic PD (iPD), and healthy volunteers (HVs). Methods: Data from five studies were combined. Variability was assessed of glucosylceramide (various isoforms), lactosylceramide (various isoforms), glucosylsphingosine, galactosylsphingosine, GCase activity (using fluorescent 4-methylumbeliferryl-β-glucoside), and GCase protein (using enzyme-linked immunosorbent assay) in plasma, PBMCs, and CSF if available, in GBA-PD, iPD, and HVs. GSLs in leukocyte subtypes were compared in HVs. Principal component analysis was used to explore global patterns in GSLs, clinical characteristics (Movement Disorder Society -Unified Parkinson's Disease Rating Scale Part 3 [MDS-UPDRS-3], Mini-Mental State Examination [MMSE], GBA1 mutation type), and participant status (GBA-PD, iPD, HVs). Results: Within-subject between-day variability ranged from 5.8% to 44.5% and was generally lower in plasma than in PBMCs. Extracellular glucosylceramide levels (plasma) were slightly higher in GBA-PD compared with both iPD and HVs, while intracellular levels were comparable. GSLs in the different matrices (plasma, PBMCs, CSF) did not correlate. Both lactosylceramide and glucosylsphingosine were more abundant in granulocytes compared with monocytes and lymphocytes. Absolute levels of GSL isoforms differed greatly. GBA1 mutation types could not be differentiated based on GSL data. Conclusions: Glucosylceramide can stably be measured over days in both plasma and PBMCs and may be used as a biomarker in clinical trials targeting GBA-PD. Glucosylsphingosine and lactosylceramide are stable in plasma but are strongly affected by leukocyte subtypes in PBMCs. GBA-PD could be differentiated from iPD and HVs, primarily based on glucosylceramide levels in plasma.
Background: Although enzalutamide (ENZ) is an effective treatment for prostate cancer (PC), development of ENZ resistance is inevitable. One mechanism for resistance is upregulation of ARV7, a splice variant of the full-length androgen receptor (FLAR) that lacks the androgen binding and hinge region domains. Nuclear translocation of FLAR requires the hinge region for coupling to tubulin or importin. ARV7 gains nuclear entry independent of the canonical pathway. FLAR has been reported to bind to Hypoxia Inducible Factor-1α (HIF1α) under normoxic conditions in the presence of dihydrotestosterone (DHT). We hypothesized that ARV7 may also heterodimerize with HIF1α in the cytoplasm, enabling nuclear localization and transcriptional activity of both transcription factors. We reported that topotecan (TPT) blocks HIF1α mRNA translation by targeting topoisomerase I (TOPO1) resident on HIF1α mRNA. We determined if ARV7 was bound to HIF1α in ENZ resistant cells and if inhibition of HIF1α translation by topotecan (TPT) could block ARV7 nuclear localization, thereby resensitizing ARV7 expressing cells to ENZ. We also evaluated if TOPO1 resided on FLAR/ARV7 mRNA, providing an additional target for TPT. Methods: 22Rv1 ENZ resistant PC cells that express FLAR and ARV7 protein were treated with the hypoxia mimetic cobalt chloride to stabilize HIF1α. Cytoplasmic and nuclear extracts from 22Rv1 cells were used for co-immunoprecipitation (Co-IP) using anit-HIF1α antibody. The effect of DHT, ENZ, and TPT on HIF1α and AR/ARV7 expression, and interactions between Topo-1 and HIF1α and AR mRNA were determined by western blot and RNA-IP. The effects of ENZ and TPT on 22Rv1 cell viability were evaluated using the XTT viability assay. Results: Topo-I RNA IP demonstrated its linkage to HIF-1α, FLAR and ARV7 mRNA. Co-IP of 22Rv1 whole cell lysates and nuclear extracts with HIF1α-specific antibodies yielded both full length AR and ARV7 protein bands on western blots under normoxic conditions in the presence of DHT and under hypoxic conditions. TPT and ENZ treatment of 22Rv1 cells under hypoxic conditions resulted in significantly reduced HIF1α, FLAR and ARV7 nuclear localization. TPT had an IC50 of 20 nM in normoxia and 91 nM in hypoxia. ENZ had an IC50 of 24.4 uM in normoxia and 388.7 uM in hypoxia. Dose-response curves for TPT / ENZ combinations at concentrations achieved clinically demonstrated synergistic activity, with combination index values of 0.54 under normoxic and 0.19 under hypoxic conditions. Conclusions: Our data indicate that Topo I resides on HIF-1α, FLAR and ARV7 mRNA, providing a potential target for TPT-mediated inhibition of their translation. Clinically achievable TPT concentrations caused HIF1α knockdown in hypoxic 22Rv1 cells in association with diminished nuclear localization of ARV7. 22Rv1 cells showed a synergistic dose-response to the combination of TPT and enzalutamide. These findings support exploration of TPT in combination with ENZ for treatment of PC. Citation Format: John P. Fruehauf, Diana C. Gomez, Leslie Spitalny, Jai-Hyun Kim. Topotecan-mediated inhibition of HIF1α/ARV7 heterodimers in 22RV1 prostate cancer cells prevents ARV7 nuclear entry, reversing enzalutamide resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4098.
Herein, we detail a novel reverse-transcription (RT) assay to directly detect chemical adducts on RNA. We optimize a fluorescence quenching assay to detect RT polymerization and employ our approach to detect N1-alkylation of inosine, an important post-transcriptional modification, using a phenylacrylamide as a model compound. We anticipate our approach can be expanded to identify novel reagents that form adducts with RNA and further explored to understand the relationship between RT processivity and natural post-transcriptional modifications in RNA.
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