Reprogramming of mesenchymal stem cells by the synovial sarcoma-associated oncogene SYT-SSX2.

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.

Immune hemolysis involving non-ABO/RhD alloantibodies following hematopoietic stem cell transplantation.

We report two cases of severe alloimmune hemolysis after hematopoietic stem cell (HSC) transplant resulting from an anti-Jk(a). The time course of hemolysis and Jk phenotypes of the donor and recipient in the cases reported suggest that the antibody was produced by donor-derived passenger lymphocytes. Retrospective analysis of the blood bank records of all allogeneic HSC transplant patients at Barnes-Jewish Hospital from 1994 to 1999 suggests that the incidence of alloimmune hemolysis due to incompatibilities involving non-ABO or RhD red cell antigens is very low, approximately 1%. In one patient, the duration of hemolysis was shortened significantly by performing red cell exchange at the first sign of intravascular hemolysis.

Evaluation of a commercial DNA enzyme immunoassay for detection of enterovirus reverse transcription-PCR products amplified from cerebrospinal fluid specimens.

We evaluated the DiaSorin DNA enzyme immunoassay (DEIA) kit for detection of enteroviral reverse transcription-PCR (RT-PCR) products amplified from cerebrospinal fluid. By use of an optical density of 0.05 as the absorbance cutoff, 35% of 198 specimens were PCR positive, whereas 16% were culture positive. DEIA was rapid and sensitive and can help implement enterovirus RT-PCR in clinical laboratories.

High-efficiency guided-mode resonance filter.

A high-efficiency guided-mode resonance reflection filter is reported. The device consists of a surface-relief photoresist grating and an underlying HfO (2) waveguide layer deposited on a fused-silica substrate. The spectral response measured with a dye-laser beam at normal incidence exhibited a peak reflectance of 98% at a wavelength of 860 nm with sideband reflectance below approximately 5% extending over the wavelength range provided by the dye (800-900 nm). At normal incidence the filter linewidth was 2.2 nm. High-efficiency double-peak resonances occurred at nonnormal incidence, with the spectral locations of the maxima vayring with the incidence angle. The filter response at various angles of incidence agreed well with the theoretically calculated reflectance curves.

A GT box element is essential for basal and cyclic adenosine 3',5'-monophosphate regulation of the human surfactant protein A2 gene in alveolar type II cells: evidence for the binding of lung nuclear factors distinct from Sp1.

The gene encoding surfactant protein-A (SP-A) is developmentally regulated in type II cells of the fetal lung. In humans there are two SP-A genes, SP-A1 and SP-A2. The SP-A2 gene is more highly regulated by cAMP and during fetal development than SP-A1. In earlier studies we determined that 296 bp of sequence flanking the 5'-end of the SP-A2 gene is sufficient to mediate high basal and cAMP-inducible reporter gene expression in primary cultures of transfected type II cells, suggesting that this region contains important cis-acting elements involved in tissue-specific and hormonal regulation of SP-A2 promoter activity. We also observed that mutagenesis of a cAMP response element (CRE)-like sequence at -242 bp (CRE(SP-A2)) greatly reduced basal and cAMP-stimulated expression in transfected type II cells. In the present study, we identified a GT box (GGGGTGGGG) at -61 bp of SP-A2 5'-flanking sequence that is highly conserved among the SP-A genes of different species. In type II cell transfection studies, we found that mutagenesis of the GT box of SP-A2 markedly reduced basal and abolished cAMP-induced reporter gene expression. Thus, CRE(SP-A2) and the GT box cooperatively interact to mediate basal and cAMP induction of SP-A2 promoter activity in type II cells. By electrophoretic mobility shift assays (EMSA), it was observed that nuclear proteins isolated from primary cultures of type II cells bound the GT box as five specific complexes. By contrast, nuclear proteins isolated from lung fibroblasts displayed notably reduced binding activity. Competition and supershift EMSA indicate that the ubiquitously expressed transcription factor Sp1, a GC box-binding protein of approximately 100 kDa, is a component of the complex of proteins that bind the GT box of SP-A2. The finding that only two of the five GT box-binding complexes were supershifted by incubation with Sp1 antibody suggests that a factor(s) in type II cell nuclear extracts that is distinct from Sp1 also interacts with the GT box. By UV cross-linking and SDS-PAGE/EMSA analysis, we have identified a approximately 55-kDa GT box-binding factor in type II cell nuclear proteins that preferentially binds the GT box of SP-A2 over the consensus Sp1 GC box sequence. This 55-kDa factor was able to bind the GT box independently of Sp1.

A CRE-like element plays an essential role in cAMP regulation of human SP-A2 gene in alveolar type II cells.

The human has two genes encoding surfactant protein-A (SP-A), termed SP-A1 and SP-A2; the SP-A2 gene is more highly regulated by cAMP and during fetal development than is SP-A1. In this study, by use of primary cultures of human type II cells transfected with fusion genes containing various amounts of SP-A2 5'-flanking DNA linked to human growth hormone (hGH) structural gene, as reporter, we found that -296 bp of SP-A2 upstream sequence is sufficient to direct high basal and cAMP-inducible expression in type II cells, but not in other cell types. By use of competitive EMSA, we observed that nuclear proteins isolated from midtrimester human fetal lung tissue bind specifically to a cAMP response element (CRE)-like sequence, TGACCTTA, at -242 bp, which we have termed CRESP-A2. Binding activity of CRESP-A2 for nuclear proteins from human fetal lung tissue before culture was manifest as two complexes of different mobilities and equivalent intensity. By contrast, upon differentiation of the human fetal lung in culture in the presence of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), the higher mobility complex was decreased to undetectable levels. By UV cross-linking analysis, using nuclear extracts from midgestation human fetal lung before culture and radiolabeled CRESP-A2 as a probe, we observed binding of proteins of approximately 50, 36, and 30 kDa. When nuclear extracts from human fetal lung cultured in the presence of DBcAMP were analyzed, binding of only the 50- and 36-kDa proteins was apparent. On the other hand, when the canonical CRE (TGACGTCA) known to bind the transcription factor CREB (M(r) approximately 43,000) was used as a probe, binding of only a approximately 43-kDa protein was evident using nuclear extracts from human fetal lung before and after culture. In type II cells transfected with an SP-A2(-296):hGH fusion gene in which CRESP-A2 was mutated, there was a marked reduction of basal and cAMP-stimulated fusion gene expression. These findings indicate that CRESP-A2 serves an important role in mediating basal and cAMP-inducible expression of the human SP-A2 gene in type II cells, that the fetal lung nuclear proteins bound to CRESP-A2 differ from those bound to the canonical palindromic CRE, and that changes in the complex of nuclear proteins bound to CRESP-A2 accompany induction of SP-A gene expression.