title: Novel Assay to Detect RNA Polymerase I ActivityIn Vivo.
abstract: This report develops an analytically validated chromogenicin situhybridization (CISH) assay using branched DNA signal amplification (RNAscope) for detecting the expression of the 5' external transcribed spacer (ETS) of the 45S ribosomal (r) RNA precursor in formalin-fixed and paraffin-embedded (FFPE) human tissues. 5'ETS/45S CISH was performed on standard clinical specimens and tissue microarrays (TMA) from untreated prostate carcinomas, high-grade prostatic intraepithelial neoplasia (PIN), and matched benign prostatic tissues. Signals were quantified using image analysis software. The 5'ETS rRNA signal was restricted to the nucleolus. The signal was markedly attenuated in cell lines and in prostate tissue slices after pharmacologic inhibition of RNA polymerase I (Pol I) using BMH-21 or actinomycin D, and by RNAi depletion of Pol I, demonstrating validity as a measure of Pol I activity. Clinical human prostate FFPE tissue sections and TMAs showed a marked increase in the signal in the presumptive precursor lesion (high-grade PIN) and invasive adenocarcinoma lesions (P= 0.0001 andP= 0.0001, respectively) compared with non-neoplastic luminal epithelium. The increase in 5'ETS rRNA signal was present throughout all Gleason scores and pathologic stages at radical prostatectomy, with no marked difference among these. This precursor rRNA assay has potential utility for detection of increased rRNA production in various tumor types and as a novel companion diagnostic for clinical trials involving Pol I inhibition.Implications:Increased rRNA production, a possible therapeutic target for multiple cancers, can be detected with a new, validated assay that also serves as a pharmacodynamic marker for Pol I inhibitors.Mol Cancer Res; 15(5); 577-84. ©2017 AACR.
authors.affiliation: Department of Pathology, Urology and Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Gunes
authors.initials: G
authors.lastname: Guner
authors.affiliation: Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Paul
authors.initials: P
authors.lastname: Sirajuddin
authors.affiliation: Department of Pathology, Urology and Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Qizhi
authors.initials: Q
authors.lastname: Zheng
authors.affiliation: Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Baoyan
authors.initials: B
authors.lastname: Bai
authors.affiliation: Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Brodie
authors.affiliation: Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Hester
authors.initials: H
authors.lastname: Liu
authors.affiliation: Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, Finland.
authors.forename: Taija
authors.initials: T
authors.lastname: Af Hällström
authors.affiliation: Department of Pathology, Urology and Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Ibrahim
authors.initials: I
authors.lastname: Kulac
authors.affiliation: Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland. ademarz@jhmi.edu mlaiho1@jhmi.edu.|Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Marikki
authors.initials: M
authors.lastname: Laiho
authors.affiliation: Department of Pathology, Urology and Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland. ademarz@jhmi.edu mlaiho1@jhmi.edu.|Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Angelo M
authors.initials: AM
authors.lastname: De Marzo
mesh_terms: D000328:Adult; D000368:Aged; D021121:Branched DNA Signal Amplification Assay / Q000379:methods*; D045744:Cell Line, Tumor; D021903:DNA, Ribosomal Spacer / Q000235:genetics*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D008297:Male; D008875:Middle Aged; D011467:Prostate / Q000378:metabolism* / Q000473:pathology; D019048:Prostatic Intraepithelial Neoplasia / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D012318:RNA Polymerase I / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000235:genetics*
chemical_list: D021903:DNA, Ribosomal Spacer; D012335:RNA, Ribosomal; D012318:RNA Polymerase I