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

doi:10.1158/1541-7786.MCR-16-0246

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

School of Medicine

Research output: Contribution to journal › Article › peer-review

6 Scopus citations

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 language	English (US)
Pages (from-to)	577-584
Number of pages	8
Journal	Molecular Cancer Research
Volume	15
Issue number	5
DOIs	https://doi.org/10.1158/1541-7786.MCR-16-0246
State	Published - May 2017

ASJC Scopus subject areas

Molecular Biology
Oncology
Cancer Research

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10.1158/1541-7786.MCR-16-0246

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@article{1a55a1a66f5743019694400ddaefae0b,

title = "Novel assay to detect RNA polymerase I activity in vivo",

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.",

author = "Gunes Guner and Paul Sirajuddin and Qizhi Zheng and Baoyan Bai and Alexandra Brodie and Hester Liu and {Af H{\"a}llstr{\"o}m}, Taija and Ibrahim Kulac and Marikki Laiho and {De Marzo}, {Angelo M.}",

note = "Funding Information: This study was supported by NIH 1R01CA172069, NIH R21CA193637, 2014 Safeway Foundation-PCF Challenge Award, Harrington Scholar-Innovator Award, Academy of Finland (288364; to M. Laiho), NIH P30 CA006973 (to M. Laiho and A. De Marzo) and 2014 Safeway Foundation-PCF Challenge Award, NIH/NCI Prostate SPORE P50CA058236, the Department of Defense Prostate Cancer Research Program, DOD Award Nos. W81XWH-10-2-0056 and W81XWH-10-2-0046 PCRP Prostate Cancer Biorepository Network (PCBN; to A. De Marzo). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Publisher Copyright: {\textcopyright} 2017 American Association for Cancer Research.",

year = "2017",

month = may,

doi = "10.1158/1541-7786.MCR-16-0246",

language = "English (US)",

volume = "15",

pages = "577--584",

journal = "Molecular Cancer Research",

issn = "1541-7786",

publisher = "American Association for Cancer Research Inc.",

number = "5",

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TY - JOUR

T1 - Novel assay to detect RNA polymerase I activity in vivo

AU - Guner, Gunes

AU - Sirajuddin, Paul

AU - Zheng, Qizhi

AU - Bai, Baoyan

AU - Brodie, Alexandra

AU - Liu, Hester

AU - Af Hällström, Taija

AU - Kulac, Ibrahim

AU - Laiho, Marikki

AU - De Marzo, Angelo M.

N1 - Funding Information: This study was supported by NIH 1R01CA172069, NIH R21CA193637, 2014 Safeway Foundation-PCF Challenge Award, Harrington Scholar-Innovator Award, Academy of Finland (288364; to M. Laiho), NIH P30 CA006973 (to M. Laiho and A. De Marzo) and 2014 Safeway Foundation-PCF Challenge Award, NIH/NCI Prostate SPORE P50CA058236, the Department of Defense Prostate Cancer Research Program, DOD Award Nos. W81XWH-10-2-0056 and W81XWH-10-2-0046 PCRP Prostate Cancer Biorepository Network (PCBN; to A. De Marzo). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Publisher Copyright: © 2017 American Association for Cancer Research.

PY - 2017/5

Y1 - 2017/5

N2 - 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.

AB - 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.

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DO - 10.1158/1541-7786.MCR-16-0246

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AN - SCOPUS:85018307405

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SP - 577

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JO - Molecular Cancer Research

JF - Molecular Cancer Research

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ER -

Novel assay to detect RNA polymerase I activity in vivo

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