Loss of MEF2D expression inhibits differentiation and contributes to oncogenesis in rhabdomyosarcoma cells.

BACKGROUND: Rhabdomyosarcoma (RMS) is a highly malignant pediatric cancer that is the most common form of soft tissue tumors in children. RMS cells have many features of skeletal muscle cells, yet do not differentiate. Thus, our studies have focused on the defects present in these cells that block myogenesis. METHODS: Protein and RNA analysis identified the loss of MEF2D in RMS cells. MEF2D was expressed in RD and RH30 cells by transient transfection and selection of stable cell lines, respectively, to demonstrate the rescue of muscle differentiation observed. A combination of techniques such as proliferation assays, scratch assays and soft agar assays were used with RH30 cells expressing MEF2D to demonstrate the loss of oncogenic growth in vitro and xenograft assays were used to confirm the loss of tumor growth in vivo. RESULTS: Here, we show that one member of the MEF2 family of proteins required for normal myogenesis, MEF2D, is largely absent in RMS cell lines representing both major subtypes of RMS as well as primary cells derived from an embryonal RMS model. We show that the down regulation of MEF2D is a major cause for the failure of RMS cells to differentiate. We find that MyoD and myogenin are bound with their dimerization partner, the E proteins, to the promoters of muscle specific genes in RMS cells. However, we cannot detect MEF2D binding at any promoter tested. We find that exogenous MEF2D expression can activate muscle specific luciferase constructs, up regulate p21 expression and increase muscle specific gene expression including the expression of myosin heavy chain, a marker for skeletal muscle differentiation. Restoring expression of MEF2D also inhibits proliferation, cell motility and anchorage independent growth in vitro. We have confirmed the inhibition of tumorigenicity by MEF2D in a tumor xenograft model, with a complete regression of tumor growth. CONCLUSIONS: Our data indicate that the oncogenic properties of RMS cells can be partially attributed to the loss of MEF2D expression and that restoration of MEF2D may represent a useful therapeutic strategy to decrease tumorigenicity

Alternative splicing of MEF2C pre-mRNA controls its activity in normal myogenesis and promotes tumorigenicity in rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Many cellular disruptions contribute to the progression of this pediatric cancer, including aberrant alternative splicing. The MEF2 family of transcription factors regulates many developmental programs, including myogenesis. MEF2 gene transcripts are subject to alternate splicing to generate protein isoforms with divergent functions. We found that MEF2Cα1 was the ubiquitously expressed isoform that exhibited no myogenic activity and that MEF2Cα2, the muscle-specific MEF2C isoform, was required for efficient differentiation. We showed that exon α in MEF2C was aberrantly alternatively spliced in RMS cells, with the ratio of α2/α1 highly down-regulated in RMS cells compared with normal myoblasts. Compared with MEF2Cα2, MEF2Cα1 interacted more strongly with and recruited HDAC5 to myogenic gene promoters to repress muscle-specific genes. Overexpression of the MEF2Cα2 isoform in RMS cells increased myogenic activity and promoted differentiation in RMS cells. We also identified a serine protein kinase, SRPK3, that was down-regulated in RMS cells and found that expression of SRPK3 promoted the splicing of the MEF2Cα2 isoform and induced differentiation. Restoration of either MEF2Cα2 or SPRK3 inhibited both proliferation and anchorage-independent growth of RMS cells. Together, our findings indicate that the alternative splicing of MEF2C plays an important role in normal myogenesis and RMS development. An improved understanding of alternative splicing events in RMS cells will potentially reveal novel therapeutic targets for RMS treatment

Equilibrium Melting Temperature of Polymorphic Poly(L-lactide) and Its Supercooling Dependence on Growth Kinetics

In this study, the isothermal crystallization process of poly(l-lactide) (PLLA) has been investigated using in situ XRD, differential scanning calorimetry (DSC), and polarized optical microscopy (POM). Linear and nonlinear extrapolation methods have been deployed to estimate the equilibrium melting temperature ( T m 0 ), which is used for analyzing the supercooling dependence of the PLLA spherulitic growth rate (G). A double-melting behavior observed for PLLA under crystallization Tc < 120 °C has been attributed to the formation of both α′ and α crystals. The T m 0 values of both α′ and α crystals have been evaluated using the linear method (172.8 °C) and nonlinear method (196.4 °C), with the nonlinear estimate being 23.6 °C higher. A discontinuity in the temperature dependence of spherulite growth rate is observed around 128.3 °C. Regime II–III transition is found to occur at 128.3 °C when T m 0 = 196.4 °C as estimated by the nonlinear extrapolation method

Continuous fabrication and assembly of spatial cell-laden fibers for a tissue-like construct via a photolithographic-based microfluidic chip

Engineering three-dimensional (3D) scaffolds with in vivo like architecture and function has shown great potential for tissue regeneration. Here we developed a facile microfluidic-based strategy for the continuous fabrication of cell-laden microfibers with hierarchically organized architecture. We show that photolithographically fabricated microfluidic devices offer a simple and reliable way to create anatomically inspired complex structures. Furthermore, the use of photo-cross-linkable methacrylated alginate allows modulation of both the mechanical properties and biological activity of the hydrogels for targeted applications. Via this approach, multilayered hollow microfibers were continuously fabricated, which can be easily assembled in situ, using 3D printing, into a larger, tissue-like construct. Importantly, this biomimetic approach promoted the development of phenotypical functions of the target tissue. As a model to engineer a complex tissue construct, osteon-like fiber was biomimetically engineered, and enhanced vasculogenic and osteogenic expression were observed in the encapsulated human umbilical cord vein endothelial cells and osteoblast-like MG63 cells respectively within the osteon fibers. The capability of this approach to create functional building blocks will be advantageous for bottom-up regeneration of complex, large tissue defects and, more broadly, will benefit a variety of applications in tissue engineering and biomedical research

High affinity binding of H3K14ac through collaboration of bromodomains 2, 4 and 5 is critical for the molecular and tumor suppressor functions of PBRM1.

Polybromo-1 (PBRM1) is an important tumor suppressor in kidney cancer. It contains six tandem bromodomains (BDs), which are specialized structures that recognize acetyl-lysine residues. While BD2 has been found to bind acetylated histone H3 lysine 14 (H3K14ac), it is not known whether other BDs collaborate with BD2 to generate strong binding to H3K14ac, and the importance of H3K14ac recognition for the molecular and tumor suppressor function of PBRM1 is also unknown. We discovered that full-length PBRM1, but not its individual BDs, strongly binds H3K14ac. BDs 2, 4, and 5 were found to collaborate to facilitate strong binding to H3K14ac. Quantitative measurement of the interactions between purified BD proteins and H3K14ac or nonacetylated peptides confirmed the tight and specific association of the former. Interestingly, while the structural integrity of BD4 was found to be required for H3K14ac recognition, the conserved acetyl-lysine binding site of BD4 was not. Furthermore, simultaneous point mutations in BDs 2, 4, and 5 prevented recognition of H3K14ac, altered promoter binding and gene expression, and caused PBRM1 to relocalize to the cytoplasm. In contrast, tumor-derived point mutations in BD2 alone lowered PBRM1\u27s affinity to H3K14ac and also disrupted promoter binding and gene expression without altering cellular localization. Finally, overexpression of PBRM1 variants containing point mutations in BDs 2, 4, and 5 or BD2 alone failed to suppress tumor growth in a xenograft model. Taken together, our study demonstrates that BDs 2, 4, and 5 of PBRM1 collaborate to generate high affinity to H3K14ac and tether PBRM1 to chromatin. Mutations in BD2 alone weaken these interactions, and this is sufficient to abolish its molecular and tumor suppressor functions

Optical lithography

Optical lithography is a photon-based technique comprised of projecting an image into a photosensitive emulsion (photoresist) coated onto a substrate such as a silicon wafer. It is the most widely used lithography process in the high volume manufacturing of nano-electronics by the semiconductor industry. Optical lithography’s ubiquitous use is a direct result of its highly parallel nature allowing vast amounts of information to be transferred very rapidly. For example, a modern leading edge lithography tool produces 150-300-mm patterned wafers per hour with 40-nm two-dimensional pattern resolution, yielding a pixel throughput of approximately 1.8T pixels/s. Continual advances in optical lithography capabilities have enabled the computing revolution over the past 50 years