ABSTRACT
Wilms' tumor gene WT1 is overexpressed in leukemia and various types of solid tumors and plays an important role in leukemogenesis and tumorigenesis. We tested apoptosis-inducing ability of short hairpin RNAs targeting exon 5 (shWTE5), exon10 (shWTE10) and 3'UTR (shWT3U) of the WT1 gene. Among the three WT1-shRNAs, since shWTE5 most effectively induced apoptosis, its ability as an apoptosis-inducing agent was intensively examined. shWTE5 induced mitochondrial damage and resultant apoptosis in five WT1-expressing solid cancer cells originated from gastric (AZ-521), lung (LU99B), ovarian (TYKnuCPr) cancers, fibrosarcoma (HT-1080) and glioblastoma (A172). Moreover, shWTE5 significantly enhanced apoptosis induced by chemotherapeutic agents, doxorubicin (DOX) and etoposide (ETP), or by death ligand TRAIL in all of the four solid tumor cells examined (HT-1080, LU99B, TYK and A172). Transduction of one each of WT1 isoforms with exon 5 [17AA(+)KTS(+) and 17AA(+)KTS(-)] prevented mitochondrial damage induced by ETP or TRAIL and inhibited apoptosis. These results showed that shWTE5 induced apoptosis through the suppression of the WT1 isoform with exon 5. Furthermore, shWTE5 increased expression of proapoptotic Bak and Bax proteins and decreased antiapoptotic Bcl-xL and Bcl-2 proteins in WT1-expressing HT-1080 cells, indicating that WT1 isoforms with exon 5 might play an antiapoptotic role through regulation of Bcl-2 family genes in solid tumor cells. The results presented here demonstrated that WT1-shRNA targeting exon 5 should serve as a potent anti-cancer agent for various types of solid tumors.
Subject(s)
Apoptosis/genetics , Genes, Wilms Tumor , Neoplasms/genetics , Neoplasms/therapy , RNA, Small Interfering/genetics , RNA, Small Interfering/therapeutic use , Antineoplastic Agents/pharmacology , Doxorubicin/pharmacology , Drug Synergism , Etoposide/pharmacology , Exons , Gene Expression Regulation, Neoplastic/drug effects , Genes, Wilms Tumor/physiology , Genes, bcl-2 , Genetic Therapy , HeLa Cells , Humans , Neoplasms/pathology , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Small Interfering/pharmacology , Signal Transduction/genetics , TNF-Related Apoptosis-Inducing Ligand/genetics , Transfection , Tumor Cells, Cultured , WT1 Proteins/antagonists & inhibitors , WT1 Proteins/metabolismABSTRACT
We obtained two novel W chromosome-linked chick genes by the use of female-male subtraction macroarrays, one of which, 2d-2F9, (recorded as AB188527 in DDBJ) did not have sufficient length (776 bp) to reveal its real form or characteristics. Hence, we obtained full-length Z-linked and W-linked 2d-2F9 genes of 2596 bp and 2589 bp respectively by the oligo-capping and RACE methods. Sequence analysis of these genes not only revealed that there is a counterpart of the W-linked 2d-2F9 gene on the Z chromosome, but also that there is a low homologous area at 5'-UTR between the W- and Z-kinked genes. Using this information, we designed a set of primers to identify sex and to select clones having the Z and W-linked gene (named 2d-2F9-Z and 2d-2F9-W), and also prepared two sets of primers for RT-PCR. These genes were found to be expressed constitutively and ubiquitously from the early embryo to the hatched chick, and they were assigned to the AAA ATP-superfamily.
