title: RNA disruption is associated with response to multiple classes of chemotherapy drugs in tumor cell lines.
abstract: BACKGROUND
  Cellular stressors and apoptosis-inducing agents have been shown to induce ribosomal RNA (rRNA) degradation in eukaryotic cells. Recently, RNA degradation in vivo was observed in patients with locally advanced breast cancer, where mid-treatment tumor RNA degradation was associated with complete tumor destruction and enhanced patient survival. However, it is not clear how widespread chemotherapy induced "RNA disruption" is, the extent to which it is associated with drug response or what the underlying mechanisms are.
  
  
  METHODS
  Ovarian (A2780, CaOV3) and breast (MDA-MB-231, MCF-7, BT474, SKBR3) cancer cell lines were treated with several cytotoxic chemotherapy drugs and total RNA was isolated. RNA was also prepared from docetaxel resistant A2780DXL and carboplatin resistant A2780CBN cells following drug exposure. Disruption of RNA was analyzed by capillary electrophoresis. Northern blotting was performed using probes complementary to the 28S and 18S rRNA to determine the origins of degradation bands. Apoptosis activation was assessed by flow cytometric monitoring of annexin-V and propidium iodide (PI) binding to cells and by measuring caspase-3 activation. The link between apoptosis and RNA degradation (disruption) was investigated using a caspase-3 inhibitor.
  
  
  RESULTS
  All chemotherapy drugs tested were capable of inducing similar RNA disruption patterns. Docetaxel treatment of the resistant A2780DXL cells and carboplatin treatment of the A2780CBN cells did not result in RNA disruption. Northern blotting indicated that two RNA disruption bands were derived from the 3'-end of the 28S rRNA. Annexin-V and PI staining of docetaxel treated cells, along with assessment of caspase-3 activation, showed concurrent initiation of apoptosis and RNA disruption, while inhibition of caspase-3 activity significantly reduced RNA disruption.
  
  
  CONCLUSIONS
  Supporting the in vivo evidence, our results demonstrate that RNA disruption is induced by multiple chemotherapy agents in cell lines from different tissues and is associated with drug response. Although present, the link between apoptosis and RNA disruption is not completely understood. Evaluation of RNA disruption is thus proposed as a novel and effective biomarker to assess response to chemotherapy drugs in vitro and in vivo.
authors.affiliation: Department of Biology, Laurentian University, Sudbury, ON, Canada.
authors.forename: Rashmi
authors.initials: R
authors.lastname: Narendrula
authors.affiliation: Department of Chemistry and Biochemistry, Laurentian University, Sudbury, ON, Canada.
authors.forename: Kyle
authors.initials: K
authors.lastname: Mispel-Beyer
authors.affiliation: Advanced Medical Research Institute of Canada, Sudbury, ON, Canada.|RNA Diagnostics Inc., Toronto, ON, Canada.
authors.forename: Baoqing
authors.initials: B
authors.lastname: Guo
authors.affiliation: Department of Biology, Laurentian University, Sudbury, ON, Canada.|Department of Chemistry and Biochemistry, Laurentian University, Sudbury, ON, Canada.|Division of Medical Sciences, Northern Ontario School of Medicine, 935 Ramsey Lake Rd., Sudbury, ON, P3E 2C6, Canada.|Advanced Medical Research Institute of Canada, Sudbury, ON, Canada.|Faculty of Medicine, Division of Oncology, University of Ottawa, Ottawa, ON, Canada.|RNA Diagnostics Inc., Toronto, ON, Canada.
authors.forename: Amadeo M
authors.initials: AM
authors.lastname: Parissenti
authors.affiliation: RNA Diagnostics Inc., Toronto, ON, Canada.
authors.forename: Laura B
authors.initials: LB
authors.lastname: Pritzker
authors.affiliation: RNA Diagnostics Inc., Toronto, ON, Canada.
authors.forename: Ken
authors.initials: K
authors.lastname: Pritzker
authors.affiliation: RNA Diagnostics Inc., Toronto, ON, Canada.
authors.forename: Twinkle
authors.initials: T
authors.lastname: Masilamani
authors.affiliation: RNA Diagnostics Inc., Toronto, ON, Canada.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Biology, Laurentian University, Sudbury, ON, Canada. clanner@nosm.ca.|Department of Chemistry and Biochemistry, Laurentian University, Sudbury, ON, Canada. clanner@nosm.ca.|Division of Medical Sciences, Northern Ontario School of Medicine, 935 Ramsey Lake Rd., Sudbury, ON, P3E 2C6, Canada. clanner@nosm.ca.
authors.forename: Carita
authors.initials: C
authors.lastname: Lannér
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics*; D016190:Carboplatin / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D000077143:Docetaxel; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D005260:Female; D006801:Humans; D061986:MCF-7 Cells; D010051:Ovarian Neoplasms / Q000188:drug therapy / Q000235:genetics*; D020871:RNA Stability / Q000187:drug effects*; D012337:RNA, Ribosomal, 18S / Q000737:chemistry* / Q000235:genetics; D012339:RNA, Ribosomal, 28S / Q000737:chemistry* / Q000235:genetics; D043823:Taxoids / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D012337:RNA, Ribosomal, 18S; D012339:RNA, Ribosomal, 28S; D043823:Taxoids; D000077143:Docetaxel; D016190:Carboplatin; D053148:Caspase 3