Ribozyme-mediated cleavage of wt1 transcripts suppresses growth of leukemia cells

Objective. The Wilms' tumor gene product (WT1) was identified as a tumor su ppressor in pediatric kidney tumors. Conversely, acute leukemias express WT 1 at a high frequency, and leukemias with high levels of WT1 expressed by l eukemic blast cells have a significantly worse prognosis, suggesting an onc ogenic function of WT1 in leukemic cells. To address this issue, we develop ed five hammerhead ribozymes (RZ1-RZ5) designed to cleave various wt1-mRNA GUC-recognition sites and thus suppress wt1 expression. Methods. Using in vitro transcribed ribozymes and truncated wt1 target RNAs as substrates, we performed in vitro cleavage assays. The sequence of two ribozymes was then cloned into the pCDNA3 expression vector containing a se lf-processing ribozyme cassette. Downregulation of wt1 due to ribozyme expr ession was analyzed in the human 293 embryonic kidney and the K562 chronic myeloid leukemia cell line by Western blotting and RT-PCR. Growth of stable transfected K562 cells was determined by proliferation analysis and IH-thy midine incorporation. Results. In vitro, the anti-wt1 ribozymes were able to recognize and cleave the target RNA in a highly sequence-specific and time-dependent manner. Th e ribozymes showed different catalytic activity. Coexpression of wt1 and th e self-processing ribozymes pRZ3 and pRZ5, respectively, resulted in a sign ificantly downregulated WT1 protein level when transiently transfected in 2 93 cells. Furthermore, stable transfection of pRZ3 and pRZ5 resulted in con siderably reduced expression of endogenous wt1 in K562 cells, correlating w ith the inhibition of cell proliferation and the induction of cell death. Conclusion. Our data suggest that anti-wt1 ribozymes area potent inhibitor of wt1 expression with possible implications for the inhibition of cell pro liferation in leukemic cells. (C) 2001 International Society for Experiment al Hematology. Published by Elsevier Science Inc.