Cleavage of the Ewing Tumour-specific EWSR1-FLI1 mRNA by Hammerhead Ribozymes

• Published in: Anticancer research Vol. 29; no. 6; pp. 1901 - 1908
• Main Authors: HÜHN, Regina, STAEGE, Martin S, HESSE, Manuela, LIEBIG, Beate, BURDACH, Stefan E. G
• Format: Journal Article
• Language: English
• Published: Attiki International Institute of Anticancer Research 01-06-2009
• Subjects: Base Sequence; Biological and medical sciences; Bone Neoplasms - genetics; Bone Neoplasms - metabolism; Bone Neoplasms - pathology; Calmodulin-Binding Proteins - genetics; Calmodulin-Binding Proteins - metabolism; Diseases of the osteoarticular system; Humans; Medical sciences; Molecular Sequence Data; Nucleic Acid Conformation; Oncogene Proteins, Fusion - genetics; Oncogene Proteins, Fusion - metabolism; Proto-Oncogene Protein c-fli-1 - genetics; Proto-Oncogene Protein c-fli-1 - metabolism; RNA, Catalytic - chemical synthesis; RNA, Catalytic - pharmacology; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Neoplasm - genetics; RNA, Neoplasm - metabolism; RNA-Binding Protein EWS; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Sarcoma, Ewing - genetics; Sarcoma, Ewing - metabolism; Sarcoma, Ewing - pathology; Sequence Homology, Nucleic Acid; Tumor Cells, Cultured; Tumors; Tumors of striated muscle and skeleton; Ewing sarcoma; Cancerology; Messenger RNA; Ribozyme; Transcription factor Fli1; Diseases of the osteoarticular system; Cleavage; ribozymes; Malignant tumor; EWSR1-FLI1; Cancer; Ewing tumours
• ISSN: 0250-7005; 1791-7530

Background: Ewing family tumours (EFT) are the second most common bone tumours in children and adolescents. In the majority
of EFT, EWSR1-FLI1 (Ewing sarcoma breakpoint region 1-Friend leukaemia virus integration 1) fusion proteins can be detected
and EWSR1-FLI1 substantially contributes to the malignant phenotype of EFT. Therefore, inactivation of EWSR1-FLI1 is an interesting
strategy for EFT therapy. Materials and Methods: A ribozyme with specificity for EWSR1-FLI1 was developed and the activity
in vitro was investigated. Synthetic RNAs corresponding to EWSR1-FLI1 were used as substrates. In addition, the total RNA
from EFT cells was used as substrate and the rapid amplification of cDNA ends method for the detection of the cleavage products
was used. Results: The ribozyme cleaved the synthetic RNA in a sequence specific manner with high efficiency in vitro. Furthermore,
the expected cleavage products were detected after digestion of the total cellular RNA with this ribozyme. A point mutation
in the catalytic centre of the ribozyme abolished enzymatic activity. Conclusion: The RNA corresponding to EWSR1-FLI1 is accessible
for ribozyme mediated inactivation and ribozymes are able to cleave EWSR1-FLI1 specific RNA in the presence of a high background
of normal cellular RNAs.