Disruption of WT1 gene expression and exon 5 splicing following cytotoxic drug treatment: antisense down-regulation of exon 5 alters target gene expression and inhibits cell survival.
Deregulated expression of the Wilms' tumor gene (WT1) has been implicated in the maintenance of a malignant phenotype in leukemias and a wide range of solid tumors through interference with normal signaling in differentiation and apoptotic pathways. Expression of high levels of WT1 is associated with poor prognosis in leukemias and breast cancer. Using real-time (Taqman) reverse transcription-PCR and RNase protection assay, we have shown up-regulation of WT1 expression following cytotoxic treatment of cells exhibiting drug resistance, a phenomenon not seen in sensitive cells. WT1 is subject to alternative splicing involving exon 5 and three amino acids (KTS) at the end of exon 9, producing four major isoforms. Exon 5 splicing was disrupted in all cell lines studied following a cytotoxic insult probably due to increased exon 5 skipping. Disruption of exon 5 splicing may be a proapoptotic signal because specific targeting of WT1 exon 5-containing transcripts using a nuclease-resistant antisense oligonucleotide (ASO) killed HL60 leukemia cells, which were resistant to an ASO targeting all four alternatively spliced transcripts simultaneously. K562 cells were sensitive to both target-specific ASOs. Gene expression profiling following treatment with WT1 exon 5-targeted antisense showed up-regulation of the known WT1 target gene, thrombospondin 1, in HL60 cells, which correlated with cell death. In addition, novel potential WT1 target genes were identified in each cell line. These studies highlight a new layer of complexity in the regulation and function of the WT1 gene product and suggest that antisense directed to WT1 exon 5 might have therapeutic potential.
Renshaw
Jane
J
Section of Paediatrics, Institute of Cancer Research, 15 Cotswold Road, Belmont, Sutton, Surrey SM2 5NG, United Kingdom. Jane.Renshaw@icr.ac.uk
Orr
Rosanne M
RM
Walton
Michael I
MI
Te Poele
Robert
R
Williams
Richard D
RD
Wancewicz
Edward V
EV
Monia
Brett P
BP
Workman
Paul
P
Pritchard-Jones
Kathryn
K
D017398:Alternative Splicing / Q000187:drug effects* / Q000235:genetics; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology; D003851:Deoxyribonucleases / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects*; D004317:Doxorubicin / Q000494:pharmacology; D005091:Exons / Q000235:genetics*; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D007938:Leukemia / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D016376:Oligonucleotides, Antisense / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D013997:Time Factors; D025721:WT1 Proteins / Q000235:genetics* / Q000378:metabolism
D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D025721:WT1 Proteins; D004317:Doxorubicin; D003851:Deoxyribonucleases; D002945:Cisplatin