title: Downregulation of Hus1 by antisense oligonucleotides enhances the sensitivity of human lung carcinoma cells to cisplatin.
abstract: BACKGROUND
  In Schizosaccharomyces pombe, Hus1 is a component of the radiation sensitive (Rad) machinery that has been identified as playing a role in DNA repair and cell cycle G2/M checkpoint control pathways. Hus1 has been shown to exist in a discrete complex with at least two Rad family members, Rad1 and Rad9. Furthermore, Hus1 is essential for checkpoint activation, since Hus1 mutants fail to arrest the cell cycle in response to DNA damage or unreplicated DNA. To establish the role and relevance of human Hus1 in cell cycle regulation, the authors applied antisense technology to selectively downregulate the expression of Hus1 mRNA.
  
  
  METHODS
  Transfection of 2'-O-methoxyethyl-modified Hus1 antisense oligoribonucleotides into human H1299 nonsmall lung carcinoma cells was performed using Lipofectin as the carrier. The authors prepared RNA from transfected cells, and levels of Hus1 expression were analyzed by real time polymerase chain reaction. The growth and viability of cells treated with Hus1 antisense oligonucleotides in the presence or absence of cisplatin were analyzed and compared to controls.
  
  
  RESULTS
  Transfection of selected Hus1 antisense oligonucleotides into p53 deficient H1299 cells resulted in significant downregulation of Hus1 mRNA, up to 80%; RNA analyses reveal a maximal Hus1 antisense activity at a concentration of 200 nM with an IC50 determined to be 90 nM. The design and transfection of oligonucleotides containing three mismatches to their corresponding antisense counterparts had no or only minor effects on Hus1 mRNA levels, showing the specificity of Hus1 mRNA downregulation. The cisplatin IC50 in untransfected H1299 cells was found to be 20 microM and could be reduced significantly to only 7 microM after transfection of a Hus1 antisense oligonucleotide.
  
  
  CONCLUSIONS
  Experiments addressing the proliferation and viability of transfected H1299 cells suggest that downregulation of Hus1 by specific antisense oligonucleotides sensitizes human cells to treatment with the DNA damaging agent cisplatin.
authors.affiliation: Department of Functional Genomics, Novartis Pharma AG, Basel, Switzerland. bernd.kinzel@pharma.novartis.com
authors.forename: Bernd
authors.initials: B
authors.lastname: Kinzel
authors.forename: Jonathan
authors.initials: J
authors.lastname: Hall
authors.forename: Francois
authors.initials: F
authors.lastname: Natt
authors.forename: Jan
authors.initials: J
authors.lastname: Weiler
authors.forename: Dalia
authors.initials: D
authors.lastname: Cohen
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000473:pathology*; D018797:Cell Cycle Proteins / Q000096:biosynthesis*; D002945:Cisplatin / Q000494:pharmacology; D004249:DNA Damage; D015536:Down-Regulation; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000473:pathology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012313:RNA; D029702:Schizosaccharomyces pombe Proteins; D013873:Thionucleotides / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D016376:Oligonucleotides, Antisense; D029702:Schizosaccharomyces pombe Proteins; D013873:Thionucleotides; C106332:hus1 protein, S pombe; D012313:RNA; D002945:Cisplatin