Novel assay to detect RNA polymerase I activity in vivo

Gunes Guner, Paul Sirajuddin, Qizhi Zheng, Baoyan Bai, Alexandra Brodie, Hester Liu, Taija Af Hällström, Ibrahim Kulac, Marikki Laiho, Angelo M. De Marzo

Research output: Contribution to journalArticlepeer-review

Abstract

This report develops an analytically validated chromogenic in situ hybridization (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 and P = 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.

Original languageEnglish (US)
Pages (from-to)577-584
Number of pages8
JournalMolecular Cancer Research
Volume15
Issue number5
DOIs
StatePublished - May 2017

ASJC Scopus subject areas

  • Molecular Biology
  • Oncology
  • Cancer Research

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