Ribozyme-Mediated Cleavage of c-fos mRNA Reduces Gene Expression of DNA Synthesis Enzymes and Metallothionein

• Published in: Proceedings of the National Academy of Sciences - PNAS 1991-12, Vol.88 (23), p.10591-10595
• Main Authors: Scanlon, K. J., Jiao, L., Funato, T., Wang, W., Tone, T., Rossi, J. J., Kashani-Sabet, M.
• Format: Article
• Language: English
• Subjects: Adipocytes; Antineoplastics; Base Sequence; Biological and medical sciences; Cell Line; Cell lines; Cell Survival - drug effects; Cisplatin - pharmacology; Cloning, Molecular; DNA; DNA Polymerase I - genetics; DNA Replication; DNA Topoisomerases, Type I - genetics; Female; fos genes; Fundamental and applied biological sciences. Psychology; Gene expression; Gene Expression Regulation, Neoplastic; Genes; Genes, fos; Humans; Messenger RNA; Metallothionein - genetics; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Ovarian Neoplasms; Plasmids; Polymerase Chain Reaction; RNA; RNA, Catalytic - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; Signal Transduction; Thymidylate Synthase - genetics; Transcription, Genetic
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.88.23.10591

The c-fos gene product Fos has been implicated in many cellular processes, including signal transduction, DNA synthesis, and resistance to antineoplastic agents. A fos ribozyme (catalytic RNA) was designed to evaluate the effects of suppressing Fos protein synthesis on expression of enzymes involved in DNA synthesis, DNA repair, and drug resistance. DNA encoding the fos ribozyme (fosRb) was cloned into the pMAMneo expression plasmid, and the resultant vector was transfected into A2780DDP cells resistant to the chemotherapeutic agent cisplatin. The parental drug-sensitive A2780S cells were transfected with the pMMV vector containing the c-fos gene. Morphological alterations were accompanied by significant changes in pharmacological sensitivity in both c-fos- and fosRb-transfected cells. pMAMneo fosRb transfectants revealed decreased c-fos gene expression, concomitant with reduced thymidylate (dTMP) synthase, DNA polymerase β, topoisomerase I, and metallothionein IIA mRNAs. In contrast, c-myc expression was elevated after fos ribozyme action. Insertion of a mutant ribozyme, mainly capable of antisense activity, into A2780DDP cells resulted in smaller reductions in c-fos gene expression and in cisplatin resistance than the active ribozyme. These studies establish a role for c-fos in drug resistance and in mediating DNA synthesis and repair processes by modulating expression of genes such as dTMP synthase, DNA polymerase β, and topoisomerase I. These studies also suggest the utility of ribozymes in the analysis of cellular gene expression.