DC-Dielectrophoresis (DC-DEP), the induced motion of the dielectric particles in a spatially non-uniform DC electric field, is applied to separate biological cells by size. The locally non-uniform electric field is generated by an insulating hurdle fabricated within a PDMS microchannel. The cells experience a negative DEP (accordingly a repulsive) force at the corners of the hurdle where the gradient of local electric-field strength is the strongest. The DC-DEP force acting on the cells is proportional to the cells' size. Thus the moving cells deviate from the streamlines and the degree of deviation is dependent on the cell size. In this paper, we demonstrated by using this method that, combined with the electroosmotic flow, mixed biological cells of a few to tens of micrometers difference in diameter can be continuously separated into different collecting wells. For separating target cells of a specific size, all that is required is to adjust the voltage outputs of the electrodes.

Complexes containing p300, but not CBP, and the nuclear proto-oncoprotein SYT were detected in confluent cultures of G1-arrested cells but not in sparse cells or during S or G2. SYT sequences constitute the N-terminal segment of a fusion oncogene product, SYT-SSX, routinely detected in synovial sarcoma, an aggressive human tumor. SYT/p300 complex formation promotes cell adhesion to a fibronectin matrix, as reflected by compromise of this process in cells expressing SYT dl mutants that retain p300 binding activity and in the primary fibroblasts of p300 but not CBP heterozygous null mice. The mechanism linking the action of SYT/p300 complexes to adhesion function is, at least in part, transcription activation-independent and results in proper activation of beta1 integrin, a major adhesion receptor.

Localization of b-catenin in the cell is a key determinant in its decision to function as a critical mediator of cell adhesion at the surface or a transcription activator in the nucleus. SYT-SSX2 is the fusion product of the chromosomal translocation, t(X;18)(p11.2;q11.2), which occurs in synovial sarcoma, a soft tissue tumor. SYT-SSX2 is known to associate with chromatin remodeling complexes and is proposed to be involved in controlling gene expression. We report that SYT-SSX2 plays a direct role in b-catenin regulation. When expressed in mammalian cells, SYT-SSX2-induced b-catenin recruitment to the nucleus. Interestingly, known target genes of canonical Wnt were not activated as a result of SYT-SSX2 expression, nor was the nuclear localization of b-catenin due to one of the signaling pathways normally implicated in this event. b-Catenin accumulation in the nucleus led to the formation of a transcriptionally active nuclear complex that contained SYT-SSX2 and b-catenin. More importantly, depletion of SYT-SSX2 in primary synovial sarcoma cells resulted in loss of nuclear b-catenin signal and a significant decrease in its signaling activity. These results unravel a novel pathway in the control of b-catenin cellular transport and strongly suggest that SYT-SSX2 contributes to tumor development, in part through b-catenin signaling.

Cell identity is determined by its gene expression programs. The ability of a cell to change its identity and produce cell types outside its lineage is achieved by the activity of transcription controllers capable of reprogramming differentiation gene networks. The synovial sarcoma (SS)-associated protein, SYT–SSX2, reprograms myogenic progenitors and human bone marrow-derived mesenchymal stem cells (BMMSCs) by dictating their commitment to a pro-neural lineage. It fulfills this function by directly targeting an extensive array of neural-specific genes as well as genes of developmental pathway mediators. Concomitantly, the ability of both myoblasts and BMMSCs to differentiate into their normal myogenic and adipogenic lineages was compromised. SS is believed to arise in mesenchymal stem cells where formation of the t(X/18) translocation product, SYT–SSX, constitutes the primary event in the cancer. SYT–SSX is therefore believed to initiate tumorigenesis in its target stem cell. The data presented here allow a glimpse at the initial events that likely occur when SYT–SSX2 is first expressed, and its dominant function in subverting the nuclear program of the stem cell, leading to its aberrant differentiation, as a first step toward transformation. In addition, we identified the fibroblast growth factor receptor gene, Fgfr2, as one occupied and upregulated by SYT–SSX2. Knockdown of FGFR2 in both BMMSCs and SS cells abrogated their growth and attenuated their neural phenotype. These results support the notion that the SYT–SSX2 nuclear function and differentiation effects are conserved throughout sarcoma development and are required for its maintenance beyond the initial phase. They also provide the stem cell regulator, FGFR2, as a promising candidate target for future SS therapy.

Synovial sarcoma (SS) is an aggressive soft tissue malignancy of children and young adults, with no effective systemic therapies. Its specific oncogene, SYT-SSX (SS18-SSX), drives sarcoma initiation and development. The exact mechanism of SYT-SSX oncogenic function remains unknown. In a SYT-SSX2 transgenic model, we show that a constitutive Wnt/β-catenin signal is aberrantly activated by SYT-SSX2, and inhibition of Wnt signaling through the genetic loss of β-catenin blocks SS tumor formation. In a combination of cell-based and SS tumor xenograft models, we show that inhibition of the Wnt cascade through co-receptor blockade and the use of small molecule CK1α activators arrests SS tumor growth. We find that upregulation of the Wnt/β-catenin cascade by SYT-SSX2 correlates with its nuclear reprogramming function. These studies reveal the central role of Wnt/β-catenin signaling in SYT-SSX2-induced sarcoma genesis, and open new venues for the development of effective SS curative agents.

A calmodulin-neomycin-resistance fusion gene was introduced into Trypanosoma brucei by electroporation, and stably transformed cell lines were obtained. In all of the transformants, the fusion gene had integrated into the host genome at the cognate locus, evidently by homologous recombination within flanking calmodulin DNA. This unusual observation distinguishes trypanosomes as the only eukaryote other than yeast known to undergo gene targeting in essentially 100% of the stable transformants. It should now be possible to systematically manipulate the trypanosome genome, directing predetermined mutations to virtually any chromosomal locus.Trypanosoma brucei are parasitic protozoa and the causative agent of sleeping sickness, a major disease in Africa. This organism exhibits many fascinating biological phenomena including antigenic variation (1, 2), polycistronic transcription (1, 3), trans-splicing of RNA (4, 5), RNA editing (6, 7), and stage-dependent mitochondrial activation (8). To better study these and other features of T. brucei, a stable transformation system involving homologous gene targeting was sought, for this would allow directed mutation of the genome as well as long-term expression of introduced DNA. Such homologous gene targeting has proven extremely valuable in numerous species to selectively alter genes of interest in the host chromosome. In yeast, virtually 100% of transfected DNA that integrates into the host genome does so by homologous recombination (9,10); other species show homologous gene targeting as well, but with lower efficiency ("'0.01%-1% of the integrants in mammalian cells; refs. 11-13). Although stable transformation of another trypanosomatid, Leishmania, has been reported (14,15), the introduced DNA remained extrachromosomal, precluding the possibility of mutating the host genome.In this paper, we present evidence for stable integrative transformation of T. brucei by a fusion gene construct, CNeoC. This construct, which contains the neomycinresistance (neo) coding region flanked by extensive 5' and 3' segments of the T. brucei calmodulin locus, was designed to favor homologous targeting (16) into the chromosomal calmodulin locus. After electroporation of procyclic cultures of T. brucei with the excised fusion gene, G418-resistant cells were obtained. DNA analysis from seven of the resultant stable cell lines (detailed in this report) and preliminary evidence from 20 additional similarly derived lines showed that in all cases the neor fusion gene was integrated into the calmodulin locus of the host genome, evidently by homologous recombination. MATERIALS AND METHODSThe CNeoC Plasmid. The CNeoC plasmid (see Fig. 1) contains the translation initiation codons ofboth the T. brucei calmodulin gene (17) and the neomycin phosphotransferase (neo') gene (18) in frame and 36 base pairs apart, the translation termination regions of both genes, and the calmodulin poly(A) addition region (19). It also includes extensive flanking calmodulin sequences, extending upstream to the EcoRI...

This study demonstrates deregulation of polycomb activity by the synovial sarcoma-associated SYT-SSX2 oncogene, also known as SS18-SSX2. Synovial sarcoma is a soft tissue cancer associated with a recurrent t(X:18) translocation event that generates one of two fusion proteins, SYT-SSX1 or SYT-SSX2. The role of the translocation products in this disease is poorly understood. We present evidence that the SYT-SSX2 fusion protein interacts with the polycomb repressive complex and modulates its gene silencing activity. SYT-SSX2 causes destabilization of the polycomb subunit Bmi1, resulting in impairment of polycomb-associated histone H2A ubiquitination and reactivation of polycomb target genes. Silencing by polycomb complexes plays a vital role in numerous physiological processes. In recent years, numerous reports have implicated gain of polycomb silencing function in several cancers. This study provides evidence that, in the appropriate context, expression of the SYT-SSX2 oncogene leads to loss of polycomb function. It challenges the notion that cancer is solely associated with an increase in polycomb function and suggests that any imbalance in polycomb activity could drive the cell toward oncogenesis. These findings provide a mechanism by which the SYT-SSX2 chimera may contribute to synovial sarcoma pathogenesis.

Synovial sarcoma is a soft tissue cancer associated with a recurrent t(X:18) translocation that generates one of two fusion proteins, SYT-SSX1 or SYT-SSX2. In this study, we demonstrate that SYT-SSX2 is a unique oncogene. Rather than confer enhanced proliferation on its target cells, SYT-SSX2 instead causes a profound alteration of their architecture. This aberrant morphology included elongation of the cell body and formation of neurite-like extensions. We also observed that cells transduced with SYT-SSX2 often repulsed one another. Notably, cell repulsion is a known component of ephrin signaling. Further analysis of SYT-SSX2-infected cells revealed significant increases in the expression and activation of Eph/ephrin pathway components. On blockade of EphB2 signaling SYT-SSX2 infectants demonstrated significant reversion of the aberrant cytoskeletal phenotype. In addition, we discovered, in parallel, that SYT-SSX2 induced stabilization of the microtubule network accompanied by accumulation of detyrosinated Glu tubulin and nocodazole resistance. Glu tubulin regulation was independent of ephrin signaling. The clinical relevance of these studies was confirmed by abundant expression of both EphB2 and Glu tubulin in SYT-SSX2-positive synovial sarcoma tissues. These results indicate that SYT-SSX2 exerts part of its oncogenic effect by altering cytoskeletal architecture in an Eph-dependent manner and cytoskeletal stability through a concurrent and distinct pathway.