BLCAP induces apoptosis in human Ewing's sarcoma cells.

Bladder cancer-associated protein (BLCAP) is a novel candidate tumor suppressor gene identified from human bladder carcinoma and highly associated with the invasion of bladder cancer. We previously reported that it also plays a key role in the tumorigenesis and metastasis of human osteosarcoma. In the present study, we constructed a recombinant encoding BLCAP cDNA. Overexpression of BLCAP resulted in growth inhibition and induced apoptosis of human TC-135 Ewing's sarcoma cells in vitro. We further investigated the caspase-3/7 activity and expressions of the fusion transcription factor Ewing's sarcoma protein-friend leukemia virus integration 1 (EWS-FLI1) and the apoptosis regulator B-cell lymphoma 2 (BCL-2). Cell apoptosis was accompanied by the down-regulated expression of EWS-FLI1 and BCL-2. Our present results suggest that BLCAP may play a role not only in regulating cell proliferation but also in coordinating apoptosis through the down-regulation of BCL-2 and EWS-FLI1 in human Ewing's sarcoma cells.

EWS-Fli1 up-regulates expression of the Aurora A and Aurora B kinases.

EWS-Fli1, a fusion gene resulting from the chromosomal translocation t(11;22, q24;q12), encodes a transcriptional activator, promotes cellular transformation, and is often found in Ewing sarcoma and primitive neuroectodermal tumor. The Aurora A and Aurora B kinases belong to a highly conserved family of serine/threonine protein kinases, are tightly regulated during the cell cycle, and are overexpressed in many carcinomas. Because the relationship between the Aurora A and/or Aurora B genes and the EWS-Fli1 fusion gene is unknown, we investigated the regulatory mechanism(s) by which Aurora kinases are controlled. Knockdown of EWS-Fli1 by small interfering RNA reduced mRNA levels not only of EWS-Fli1 but also of Aurora A and Aurora B. Luciferase assay using Aurora A and Aurora B promoters showed up-regulated activities compared with those of an empty vector. Experiments with deletion and point mutants showed positive regulatory Ets-binding sites located -84 and -71 bp upstream of the transcription initiation sites in Aurora A and Aurora B, respectively. Moreover, chromatin immunoprecipitation assay revealed that EWS-Fli1 gene products interact with both the Aurora A and Aurora B promoters. These results strongly suggest that the mitotic kinases Aurora A and Aurora B are regulated by EWS-Fli1 fusion protein in Ewing sarcoma cells.

Mu-calpain is involved in the regulation of TNF-alpha-induced matrix metalloproteinase-3 release in a rheumatoid synovial cell line.

Calpain is secreted by intra-articular synovial cells and degrades the main components of cartilage matrix proteins, proteoglycan, and collagen, causing cartilage destruction. Matrix metalloproteinase-3 (MMP-3) has also been detected in synovial fluid and serum, and is involved in the development and progression of rheumatoid arthritis by degradation of the extracellular matrix and cartilage destruction. To investigate the relationship between calpain and MMP-3 in rheumatic inflammation, we utilized the rheumatic synovial cell line, MH7A. Tumor necrosis factor (TNF-alpha) stimulation-induced increased expression of mu-calpain, m-calpain, and MMP-3 in these cells, as well as the release of calpain and MMP-3 into the culture medium. The calpain inhibitors, ALLN (calpain inhibitor I) and calpeptin, did not affect the intracellular expression of MMP-3, but reduced the secretion of MMP-3 in a concentration-dependent manner. Down-regulation of mu- but not m-calpain by small interfering RNAs abolished TNF-alpha-induced MMP-3 release from the synovial cells. These findings suggest that calpain, particularly mu-calpain, regulates MMP-3 release by rheumatic synovial cells, in addition to exerting its own degradative action on cartilage.

Inhibition of platelet-derived growth factor-induced cell growth signaling by a short interfering RNA for EWS-Fli1 via down-regulation of phospholipase D2 in Ewing sarcoma cells.

EWS-Fli1, a fusion gene resulting from a chromosomal translocation t(11;22, q24;q12) and found in Ewing sarcoma and primitive neuroectodermal tumors, encodes a transcriptional activator and promotes cellular transformation. However, the precise biological functions of its products remain unknown. To investigate the role of EWS-Fli1 in cell growth signaling, we transfected Ewing sarcoma TC-135 cells with short interfering RNAs for EWS-Fli1. EWS-Fli1 knockdown reduced cell growth and platelet-derived growth factor (PDGF)-BB-induced activation of the growth signaling enzymes. Interestingly, phospholipase D2 (but not the PDGF-BB receptor) showed marked down-regulation in the EWS-Fli1-knocked down TC-135 cells compared with the control cells. In Ewing sarcoma TC-135 cells, the PDGF-BB-induced phosphorylation of growth signaling involving extracellular signal-regulated kinase, Akt, p70S6K, and the expression of cyclin D3 were markedly inhibited by transfection with short interfering RNA phospholipase (PL)-D2. The PDGF-BB-induced activation of growth signaling was also suppressed by 1-butanol, which prevents the production of phosphatidic acid by phospholipase D (but not by t-butyl alcohol), thereby implicating PLD2 in PDGF-BB-mediated signaling in TC-135 cells. These results suggest that EWS-Fli1 may play a role in the regulation of tumor proliferation-signaling enzymes via PLD2 expression in Ewing sarcoma cells.

Small interfering RNAs expressed from a Pol III promoter suppress the EWS/Fli-1 transcript in an Ewing sarcoma cell line.

The EWS/Fli-1 fusion gene encodes an oncogenic fusion protein. The fusion is a product of the translocation t(11;22) (q24;q12), which is detected in 85% of Ewing sarcoma and primitive neuroectodermal tumor cells. Utilizing intracellularly expressed 21- to 23-nucleotide small interfering RNAs (siRNAs) targeting the EWS/Fli-1 fusion transcript in an Ewing sarcoma cell line, we achieved a greater than 80% reduction in the EWS/Fli-1 transcript. The reduction in transcript levels was accompanied by growth inhibition of an Ewing cell line. In addition to quantitating the reduction of the fusion transcript, we carefully monitored reduction of the endogenous EWS and Fli-1 mRNAs as well. One of the two siRNAs targeted to the fusion transcript also partially downregulated the Fli-1 mRNA, further potentiating the growth inhibition. These results highlight both the power of siRNAs and the potential side reactions that need to be carefully monitored. In addition, these results provide the first demonstration of expressed siRNAs downregulating an oncogenic fusion transcript. The results and observations from these studies should prove useful in targeting other fusion transcripts characteristic of sarcomas and erythroleukemias.

Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells.

RNA interference (RNAi) is the process of sequence-specific, posttranscriptional gene silencing in animals and plants initiated by double-stranded (ds) RNA that is homologous to the silenced gene. This technology has usually involved injection or transfection of dsRNA in model nonvertebrate organisms. The longer dsRNAs are processed into short (19 25 nucleotides) small interfering RNAs (siRNAs) by a ribonucleotide protein complex that includes an RNAse III related nuclease (Dicer), a helicase family member, and possibly a kinase and an RNA-dependent RNA polymerase (RdRP). In mammalian cells it is known that dsRNA 30 base pairs or longer can trigger interferon responses that are intrinsically sequence-nonspecific, thus limiting the application of RNAi as an experimental and therapeutic agent. Duplexes of 21-nucleotide siRNAs with short 3' overhangs, however, can mediate RNAi in a sequence-specific manner in cultured mammalian cells. One limitation in the use of siRNA as a therapeutic reagent in vertebrate cells is that short, highly defined RNAs need to be delivered to target cells--a feat thus far only accomplished by the use of synthetic, duplex RNAs delivered exogenously to cells. In this report, we describe a mammalian Pol III promoter system capable of expressing functional double-stranded siRNAs following transfection into human cells. In the case of the 293 cells cotransfected with the HIV-1 pNL4-3 proviral DNA and the siRNA-producing constructs, we were able to achieve up to 4 logs of inhibition of expression from the HIV-1 DNA.