4-(5-benzo(1-3)dioxol-5-yl-4-pyridin-2-yl-1h-imidazol-2-yl)benzamide and Teratoma

Atypical teratoid/rhabdoid tumors (ATRT) are highly malignant brain tumors arising in young children. The majority of ATRT is characterized by inactivation of the chromatin remodeling complex member SMARCB1 (INI1/hSNF5). Little is known, however, on downstream pathways involved in the detrimental effects of SMARCB1 deficiency which might also represent targets for treatment. Using Drosophila melanogaster and the Gal4-UAS system, modifier screens were performed in order to identify the role of SMAD dependent signaling in the lethal phenotype associated with knockdown of snr1, the fly homolog of SMARCB1. Expression and functional role of human homologs was next investigated in ATRT tumor samples and SMARCB1-deficient rhabdoid tumor cells. The lethal phenotype associated with snr1 knockdown in Drosophila melanogaster could be shifted to later stages of development upon additional knockdown of several decapentaplegic pathway members including Smox, and Med. Similarly, the transforming growth factor beta (TGFbeta) receptor type I kinase inhibitor SB431542 ameliorated the detrimental effect of snr1 knockdown in the fruit fly. Examination of homologs of candidate decapentaplegic pathway members in human SMARCB1-deficent ATRT samples revealed SMAD3 and SMAD6 to be over-expressed. In SMARCB1-deficent rhabdoid tumor cells, siRNA-mediated silencing of SMAD3 or SMAD6 expression reduced TGFbeta signaling activity and resulted in decreased proliferation. Similar results were obtained upon pharmacological inhibition of TGFbeta signaling using SB431542. Our data suggest that SMAD dependent signaling is involved in the detrimental effects of SMARCB1-deficiency and provide a rationale for the investigation of TGFbeta targeted treatments in ATRT.

Topics: Animals; Benzamides; Dioxoles; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Female; Humans; Male; Rhabdoid Tumor; RNA, Messenger; Signal Transduction; Smad Proteins; Smad3 Protein; Smad6 Protein; SMARCB1 Protein; Teratoma; Transcription Factors; Transforming Growth Factor beta

Human somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by ectopic expression of key transcription factors. iPSCs have been generated from a variety of cell types. However, iPSC induction from human myoblasts has not yet been reported. Human primary skeletal myoblasts can be cultured from diagnostic muscle biopsy specimens, and thousands of lines are frozen and stored in biobanks, and are a valuable source for iPSC-based etiological and pathogenic studies. Our aim was to generate iPSCs from human skeletal myoblasts enriched from muscle biopsy samples. We used retro- or Sendai virus vector-mediated reprogramming of enriched human myoblasts from 7 donors. We show that stable iPSC lines can be generated from human myoblasts at efficiency similar to that of fibroblasts when appropriate media is used, and the efficiency of the feeder-free iPSC generation can be significantly improved by inhibitors of histone deacetylase (sodium butyrate) and TGF-β signaling (SB431542).

Topics: Adult; Animals; Antigens, Differentiation; Benzamides; Butyric Acid; Cell Culture Techniques; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Dioxoles; Female; Gene Silencing; Histone Deacetylase Inhibitors; Humans; Induced Pluripotent Stem Cells; Infant; Infant, Newborn; Male; Mice; Mice, Nude; Middle Aged; Muscle, Skeletal; Myoblasts, Skeletal; Retroviridae; Sendai virus; Signal Transduction; Teratoma; Transduction, Genetic; Transforming Growth Factor beta; Young Adult

Embryonic stem (ES) cells are self-renewing cells that maintain pluripotency to differentiate into all types of cells. Because of their potential to provide a variety of tissues for use in regenerative medicine, there is great interest in the identification of growth factors that govern these unique properties of ES cells. However, the signaling pathways controlling ES cell proliferation remain largely unknown. Since transforming growth factor beta (TGFbeta) superfamily members have been implicated in the processes of early embryogenesis, we investigated their roles in ES cell self-renewal. Inhibition of activin-Nodal-TGFbeta signaling by Smad7 or SB-431542 dramatically decreased ES cell proliferation without decreasing ES pluripotency. By contrast, inhibition of bone morphogenetic protein (BMP) signaling by Smad6 did not exhibit such effects, suggesting that activin-Nodal-TGFbeta signaling, but not BMP signaling, is indispensable for ES cell propagation. In serum-free culture, supplementation of recombinant activin or Nodal, but not TGFbeta or BMP, significantly enhanced ES cell propagation without affecting pluripotency. We also found that activin-Nodal signaling was constitutively activated in an autocrine fashion in serum-free cultured ES cells, and that inhibition of such endogenous signaling by SB-431542 decreased ES cell propagation in serum-free conditions. These findings suggest that endogenously activated autocrine loops of activin-Nodal signaling promote ES cell self-renewal.

Topics: Activins; Animals; Autocrine Communication; Benzamides; Cell Division; Chimera; Culture Media, Serum-Free; Dioxoles; Embryonic Stem Cells; Mice; Mice, Inbred C57BL; Mice, Nude; Nodal Protein; Protein Kinase Inhibitors; Signal Transduction; Smad6 Protein; Smad7 Protein; Teratoma; Transforming Growth Factor beta; Tumor Cells, Cultured