title: Inhibition of retinoblastoma tumor suppressor activity by RNA interference in lung cancer lines.
abstract: BACKGROUND
  Inactivation of retinoblastoma (RB) tumor suppressor function occurs frequently in lung cancer. Short-hairpin RNA can be constructed to target specific sequences and efficiently knock down protein expression. We developed a short-hairpin RNA approach to specifically target Rb in lung cancer cells to determine the influence of RB knockdown on proliferation.
  
  
  METHODS
  NCI-H520 human lung cancer cells (wild-type Rb) were transfected with pMSCVpuro-Rb3C, a plasmid containing a short-hairpin sequence targeted to human Rb. Transfectants harboring the construct were selected with puromycin. Loss of RB expression in selected cell populations was determined by immunoblotting. Proliferating cells were counted to establish growth rates. Retinoblastoma-proficient and RB-deficient tumor growth was monitored in nude mice.
  
  
  RESULTS
  Transfection with pMSCVpuro-Rb3C dramatically diminished RB expression and led to aberrant expression of RB-regulated genes. Cells harboring pMSCVpuro-Rb3C grew at an increased rate compared with control cells: 480.6 +/- 37.7 versus 159.4 +/- 36.2 (relative cell count at 12 days). Tumor growth in nude mice also increased with RB knockdown compared with control mice: 135.2 +/- 73.6 mm3 versus 40.0 +/- 17.0 mm3 (tumor volume at 10 days).
  
  
  CONCLUSIONS
  Inhibition of RB expression is efficiently achieved in lung cancer cells with short-hairpin RNA. Genetic targets of RB are deregulated with RB knockdown. Retinoblastoma depletion increases growth in vitro and in murine xenografts. These studies indicate that even in the context of an established tumor cell line, RB limits tumorigenic proliferation. Additionally, this model will serve as an ideal system to evaluate the role of RB activity on therapeutic response.
authors.affiliation: Division of Thoracic Surgery, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0558, USA. michael.reed@uc.edu
authors.forename: Michael F
authors.initials: MF
authors.lastname: Reed
authors.forename: William A
authors.initials: WA
authors.lastname: Zagorski
authors.forename: John A
authors.initials: JA
authors.lastname: Howington
authors.forename: Jack T
authors.initials: JT
authors.lastname: Zilfou
authors.forename: Erik S
authors.initials: ES
authors.lastname: Knudsen
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000096:biosynthesis / Q000235:genetics; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000473:pathology*; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000235:genetics; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor / Q000166:cytology / Q000187:drug effects / Q000637:transplantation; D004250:DNA Topoisomerases, Type II / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects; D005260:Female; D020869:Gene Expression Profiling; D016161:Genes, Retinoblastoma; D018816:Genes, cdc; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D009368:Neoplasm Transplantation; D020411:Oligonucleotide Array Sequence Analysis; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology*; D016160:Retinoblastoma Protein / Q000037:antagonists & inhibitors* / Q000235:genetics; D013940:Thymidylate Synthase / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
chemical_list: D000951:Antigens, Neoplasm; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; D016160:Retinoblastoma Protein; D013940:Thymidylate Synthase; D004250:DNA Topoisomerases, Type II