title: Inhibition of tumor growth by ribozyme-mediated suppression of aberrant epidermal growth factor receptor gene expression.
abstract: BACKGROUND
  Amplification and rearrangement of the epidermal growth factor receptor (EGFR) gene is frequently associated with malignant gliomas. One type of EGFR mutation in primary gliomas results in overexpression of an aberrant EGFR messenger RNA (mRNA) that lacks sequences of exons II through VI of the human EGFR gene. We observed that the aberrantly spliced EGFR mRNA contains a ribozyme cleavable sequence (5'-AAG GUA AUU-3') created by the joining of EGFR exon I to exon VII. We hypothesized that an appropriately designed ribozyme RNA could mediate site-specific cleavage of the aberrant EGFR mRNA and reduce the growth of aberrant EGFR-producing tumor cells.
  
  
  METHODS
  We synthesized aberrant EGFR mRNA substrates and a sequence-specific hammerhead ribozyme (abEGFR-rib) to examine the ribozyme's activity in vitro. We also constructed an abEGFR-rib plasmid and introduced it into ERM5-1 cells, which are murine NIH3T3 cells transfected to express an aberrant EGFR complementary DNA. We measured the growth potential of the cotransfected cells in culture and in nude mice.
  
  
  RESULTS
  The synthesized abEGFR-rib efficiently and specifically cleaved aberrant EGFR mRNA substrates in vitro. Expression of the transfected abEGFR-rib suppressed expression of aberrant EGFR mRNA in ERM5-1 cells and reduced the growth of tumors formed by the cotransfected cells in nude mice. Finally, the incorporation of bromodeoxyuridine, a measure of mitotic activity, was also decreased in abEGFR-rib-producing ERM5-1 cells in vivo.
  
  
  CONCLUSIONS
  Ribozymes targeted to aberrant EGFR mRNA can inhibit the growth of tumors formed by cells that express this mRNA.
authors.affiliation: Department of Pathology, Tokai University School of Medicine, Isehara, Kanagawa, Japan. yamazaki@is.icc.u-tokai.ac.jp
authors.forename: H
authors.initials: H
authors.lastname: Yamazaki
authors.forename: H
authors.initials: H
authors.lastname: Kijima
authors.forename: Y
authors.initials: Y
authors.lastname: Ohnishi
authors.forename: Y
authors.initials: Y
authors.lastname: Abe
authors.forename: Y
authors.initials: Y
authors.lastname: Oshika
authors.forename: T
authors.initials: T
authors.lastname: Tsuchida
authors.forename: T
authors.initials: T
authors.lastname: Tokunaga
authors.forename: A
authors.initials: A
authors.lastname: Tsugu
authors.forename: Y
authors.initials: Y
authors.lastname: Ueyama
authors.forename: N
authors.initials: N
authors.lastname: Tamaoki
authors.forename: M
authors.initials: M
authors.lastname: Nakamura
mesh_terms: D000818:Animals; D002869:Chromosome Aberrations; D015536:Down-Regulation; D066246:ErbB Receptors / Q000096:biosynthesis* / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D005910:Glioma / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D012313:RNA; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000032:analysis; D014407:Tumor Cells, Cultured
chemical_list: C034477:RNA primers; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D012313:RNA; D066246:ErbB Receptors