Inhibition of tumor growth by ribozyme-mediated suppression of aberrant epidermal growth factor receptor gene expression.

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. CONCLUSION Ribozymes targeted to aberrant EGFR mRNA can inhibit the growth of tumors formed by cells that express this mRNA.

[1]  C. Harris,et al.  Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[2]  S Amerini,et al.  Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. , 1994, The Journal of clinical investigation.

[3]  M. Ernst,et al.  Nitric oxide synthase: MRNA expression of different isoforms in human monocytes/macrophages , 1994, European journal of immunology.

[4]  A. Feinberg,et al.  A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. , 1983, Analytical biochemistry.

[5]  V Villiotou,et al.  Nitric oxide, peroxynitrite and nitroso-compounds formation by ultraviolet A (UVA) irradiated human squamous cell carcinoma: potential role of nitric oxide in cancer prognosis. , 1995, Anticancer research.

[6]  L. Kedes,et al.  A human beta-actin expression vector system directs high-level accumulation of antisense transcripts. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[7]  J. M. Buzayan,et al.  Non-enzymatic cleavage and ligation of RNAs complementary to a plant virus satellite RNA , 1986, Nature.

[8]  J. Rossi,et al.  Ribozymes as potential anti-HIV-1 therapeutic agents. , 1990, Science.

[9]  P. Humphrey,et al.  Structural alterations of the epidermal growth factor receptor gene in human gliomas. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[10]  須川 典亮.  Identical splicing of aberrant epidermal growth factor receptor transcripts from amplified rearranged genes in human glioblastomas , 1992 .

[11]  K. Aldape,et al.  Expression of nitric oxide synthase in human central nervous system tumors. , 1995, Cancer research.

[12]  B. Achinstein,et al.  Journal of the National Cancer Institute, Vol. 29, 1962: Action of bacterial polysaccharide on tumors. II. Damage of sarcoma 37 by serum of mice treated with Serratia marcescens polysaccharide, and induced tolerance. , 2009, Nutrition reviews.

[13]  H. Kijima,et al.  Tissue-specific expression of an anti-ras ribozyme inhibits proliferation of human malignant melanoma cells. , 1996, Nucleic acids research.

[14]  O. Uhlenbeck.  A small catalytic oligoribonucleotide , 1987, Nature.

[15]  Y. Takebe,et al.  Importance of independence in ribozyme reactions: kinetic behavior of trimmed and of simply connected multiple ribozymes with potential activity against human immunodeficiency virus. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[16]  D. Powe,et al.  The tumour stroma of oral squamous cell carcinomas show increased vascularity compared with adjacent host tissue. , 1997, British Journal of Cancer.

[17]  L. Kedes,et al.  Evolution of the functional human beta-actin gene and its multi-pseudogene family: conservation of noncoding regions and chromosomal dispersion of pseudogenes , 1985, Molecular and cellular biology.

[18]  W. Cavenee,et al.  A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[19]  H. Okayama,et al.  High-efficiency transformation of mammalian cells by plasmid DNA. , 1987, Molecular and cellular biology.

[20]  T. Cech,et al.  In vitro splicing of the ribosomal RNA precursor of tetrahymena: Involvement of a guanosine nucleotide in the excision of the intervening sequence , 1981, Cell.

[21]  S. Erzurum,et al.  Continuous nitric oxide synthesis by inducible nitric oxide synthase in normal human airway epithelium in vivo. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[22]  A. Franchi,et al.  Prediction of occult neck metastases in laryngeal carcinoma: role of proliferating cell nuclear antigen, MIB-1, and E-cadherin immunohistochemical determination. , 1996, Clinical cancer research : an official journal of the American Association for Cancer Research.

[23]  W. Gerlach,et al.  Simple RNA enzymes with new and highly specific endoribonuclease activities , 1988, Nature.

[24]  Y. Ozeki,et al.  Vascular endothelial growth factor expression in primary esophageal squamous cell carcinoma , 1997, Cancer.

[25]  M. Shibuya,et al.  Amplification of the structurally and functionally altered epidermal growth factor receptor gene (c-erbB) in human brain tumors , 1988, Molecular and cellular biology.

[26]  R. Schrier.  Renal and Electrolyte Disorders , 1976 .

[27]  H. Granger,et al.  Nitric oxide promotes proliferation and plasminogen activator production by coronary venular endothelium through endogenous bFGF. , 1997, Circulation research.

[28]  T. Ooi,et al.  The erbB gene of avian erythroblastosis virus is a member of the src gene family , 1983, Cell.

[29]  J. Forfar.  Normal and abnormal calcium, phosphorus and magnesium metabolism in the perinatal period. , 1976, Clinics in endocrinology and metabolism.

[30]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[31]  P. Gullino,et al.  Angiogenesis and neoplastic progression in vitro. , 1982, Journal of the National Cancer Institute.

[32]  J. Isner,et al.  Reciprocal relation between VEGF and NO in the regulation of endothelial integrity , 1997, Nature Medicine.

[33]  J. Folkman,et al.  Tumor angiogenesis and metastasis--correlation in invasive breast carcinoma. , 1991, The New England journal of medicine.

[34]  H. Granger,et al.  Nitric Oxide Is an Upstream Signal of Vascular Endothelial Growth Factor-induced Extracellular Signal-regulated Kinase½ Activation in Postcapillary Endothelium* , 1998, The Journal of Biological Chemistry.

[35]  T. Sugimura,et al.  Increased expression of inducible and endothelial constitutive nitric oxide synthases in rat colon tumors induced by azoxymethane. , 1997, Cancer research.

[36]  P. Humphrey,et al.  Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[37]  K. Dameron,et al.  Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1. , 1994, Science.

[38]  G. Tozer,et al.  Inhibition of nitric oxide synthase induces a selective reduction in tumor blood flow that is reversible with L-arginine. , 1997, Cancer research.

[39]  C. James,et al.  Amplified and rearranged epidermal growth factor receptor genes in human glioblastomas reveal deletions of sequences encoding portions of the N- and/or C-terminal tails. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[40]  A. Ullrich,et al.  Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences , 1984, Nature.

[41]  J. Rossi,et al.  Ribozyme-mediated inhibition of bcr-abl gene expression in a Philadelphia chromosome-positive cell line. , 1993, Blood.

[42]  I. Fidler,et al.  Destruction of bystander cells by tumor cells transfected with inducible nitric oxide (NO) synthase gene. , 1997, Journal of the National Cancer Institute.

[43]  L. Ignarro.  Signal transduction mechanisms involving nitric oxide. , 1991, Biochemical pharmacology.

[44]  J. M. Buzayan,et al.  Autolytic Processing of Dimeric Plant Virus Satellite RNA , 1986, Science.

[45]  P. Gullino,et al.  Gangliosides promote the angiogenic response. , 1989, Laboratory investigation; a journal of technical methods and pathology.

[46]  I. Hart,et al.  Inhibitors of nitric oxide synthase selectively reduce flow in tumour‐associated neovasculature , 1992, British journal of pharmacology.

[47]  F. Studier,et al.  Cloning and expression of the gene for bacteriophage T7 RNA polymerase. , 1984, Proceedings of the National Academy of Sciences of the United States of America.