Inhibition Of Myogenesis Research Articles

Toxoplasma gondiidown modulates cadherin expression in skeletal muscle cells inhibiting myogenesis

BackgroundToxoplasma gondii belongs to a large and diverse group of obligate intracellular parasitic protozoa. Primary culture of mice skeletal muscle cells (SkMC) was employed as a model for experimental toxoplasmosis studies. The myogenesis of SkMC was reproduced in vitro and the ability of T. gondii tachyzoite forms to infect myoblasts and myotubes and its influence on SkMC myogenesis were analyzed.ResultsIn this study we show that, after 24 h of interaction, myoblasts (61%) were more infected with T. gondii than myotubes (38%) and inhibition of myogenesis was about 75%. The role of adhesion molecules such as cadherin in this event was investigated. First, we demonstrate that cadherin localization was restricted to the contact areas between myocytes/myocytes and myocytes/myotubes during the myogenesis process. Immunofluorescence and immunoblotting analysis of parasite-host cell interaction showed a 54% reduction in cadherin expression at 24 h of infection. Concomitantly, a reduction in M-cadherin mRNA levels was observed after 3 and 24 h of T. gondii-host cell interaction.ConclusionsThese data suggest that T. gondii is able to down regulate M-cadherin expression, leading to molecular modifications in the host cell surface that interfere with membrane fusion and consequently affect the myogenesis process.

Abstract 4786: A role for increased Fra-1 expression in cachexia: Novel mechanisms of increased apoptosis in C2C12 myotubes

Abstract Over 60% of patients with advanced cancer have cancer-induced cachexia, and the prevalence of weight loss reaches 86% in end-stage cancer. While cachexia is directly responsible for approximately 25% of cancer deaths and is inversely correlated with survival time, current treatments are limited and primarily palliative. Tumor necrosis factor (TNFα) is elevated in cancer patients, and is strongly associated with cachexia as it produces pleiotropic cachectic effects including apoptosis, inhibition of myogenesis and increased protein degradation. These effects are mediated in part by the pro-inflammatory transcription factor nuclear factor kappa B (NFκB) which upregulates components of the ubiquitin-proteasome pathway, and inhibits muscle cell differentiation through post-translational suppression of the muscle specific transcription factor, myogenic determination factor 1. We believe a critical role of AP-1 in cancer cachexia precedes NFκB-activated proteasome degradation. AP-1, and more specifically a switch to Jun:Fra-1 heterodimers, induces caspase 3 activity which is necessary to cleave large actomyosin complexes prior to further degradation by the ubiquitin-proteasome system. In C2C12 mouse muscle cells treated with TNFα, we see increased expression of the AP-1 subunits cJun, JunB and Fra-1 along with the expected decrease in myosin heavy chain (MyHC) expression. Inhibiting the mitogen activated protein kinase (MAPK) pathway with U0126 in TNFα-treated cells reduces the expression of these AP-1 subunits and partially restores expression of MyHC. When we overexpress the Fra-1 subunit in C2C12 cells, we see increased mRNA expression of FAS and caspases 1, 8 and 11 (ortholog of human caspase 4). Caspase 11-mediated activation of caspase 1 appears to activate IL-1β, a catabolism-inducing cytokine implicated in cachexia. Downstream of Fra-1 expression, we also see increased caspase 3 activity and an increase in the number of TUNEL-positive apoptotic cells. We hypothesize that TNFα signaling through the MAPK pathway leads to phosphorylation-dependent stabilization of Fra-1, and leads to a switch to Jun:Fra-1 heterodimer composition which, in turn, transcriptionally up-regulates FAS and caspases 1, 8 and 11. We think increased expression of FAS, and caspases 8 and 11 lead to FAS-mediated apoptosis and increased caspase 3 activity. We used high-throughput screening to identify AP-1 inhibitors, and then tested these inhibitors for their ability to restore MyHC expression in TNFα-treated cells. We have identified a novel compound, LS474, which specifically inhibits Fra-1 expression and restores MyHC expression. This suggests that the Fra-1 subunit of AP-1 plays an important role in mediating cachexia and is a good therapeutic target in the treatment of cancer cachexia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4786. doi:10.1158/1538-7445.AM2011-4786

An evolutionarily acquired genotoxic response discriminates MyoD from Myf5, and differentially regulates hypaxial and epaxial myogenesis

Despite having distinct expression patterns and phenotypes in mutant mice, the myogenic regulatory factors Myf5 and MyoD have been considered to be functionally equivalent. Here, we report that these factors have a different response to DNA damage, due to the presence in MyoD and absence in Myf5 of a consensus site for Abl-mediated tyrosine phosphorylation that inhibits MyoD activity in response to DNA damage. Genotoxins failed to repress skeletal myogenesis in MyoD-null embryos; reintroduction of wild-type MyoD, but not mutant Abl phosphorylation-resistant MyoD, restored the DNA-damage-dependent inhibition of muscle differentiation. Conversely, introduction of the Abl-responsive phosphorylation motif converts Myf5 into a DNA-damage-sensitive transcription factor. Gene-dosage-dependent reduction of Abl kinase activity in MyoD-expressing cells attenuated the DNA-damage-dependent inhibition of myogenesis. The presence of a DNA-damage-responsive phosphorylation motif in vertebrate, but not in invertebrate MyoD suggests an evolved response to environmental stress, originated from basic helix-loop-helix gene duplication in vertebrate myogenesis.

Inhibition of myotube formation by paraquat in the myoblast cell line C2C12

Paraquat (PQ) is one of the most frequently used pesticides in worldwide. In most countries, PQ is used without restrictions. To investigate the effect of PQ on myogenesis, cultures of C2C12, a useful model to study differentiation of myoblasts into myotubes, were exposed to various concentrations of PQ. Myotube formation did not occur in the presence of 50 µM PQ. Although cell death was not observed at this concentration, growth inhibition was evident in the growth medium. Production of myosin heavy chain, a myogenesis marker protein, decreased dose dependently with the concentration of PQ, which was added to the C2C12 cell culture during differentiation. Inhibition of myogenesis by PQ was not reversed by the addition of ascorbic acid. These results show that PQ is a strong inhibitor of muscle differentiation in vitro.

RNA‐binding motif protein 24 regulates myogenin expression and promotes myogenic differentiation

The formation of muscle fibers involves sequential expression of many proteins that regulate key steps during myoblast-to-myotube transition. Myogenin is a major player in the initiation and maintenance of myogenic differentiation in a mouse myoblast cell line, C2C12. RNA-binding proteins bind to specific target RNA sequences and regulate gene expression in a post-transcriptional manner. This study demonstrates that RNA-binding motif protein 24 (Rbm24) interacts with the 3'-untranslated region of myogenin mRNA and affects its half-life in C2C12 myogenesis. Knockdown of Rbm24 expression by RNA interference significantly decreased myogenin expression associated with the inhibition of myogenesis. In contrast, the overexpression of Rbm24 by stable transfection of a plasmid increased myogenin expression and had a positive effect on myogenic differentiation. Ectopic expression of myogenin was also able to restore myogenic differentiation in Rbm24-knockdown cells. Together, our results suggest that Rbm24 binds to myogenin mRNA and regulates its stability in C2C12 cells. Rbm24 plays a crucial role in myogenic differentiation at least in part through a myogenin-dependent post-transcriptional regulatory pathway.

Dual Roles of Smad Proteins in the Conversion from Myoblasts to Osteoblastic Cells by Bone Morphogenetic Proteins

Bone morphogenetic proteins (BMPs) induce ectopic bone formation in muscle tissue in vivo and convert myoblasts such that they differentiate into osteoblastic cells in vitro. We report here that constitutively active Smad1 induced osteoblastic differentiation of C2C12 myoblasts in cooperation with Smad4 or Runx2. In floxed Smad4 mice-derived cells, Smad4 ablation partially suppressed BMP-4-induced osteoblast differentiation. In contrast, the BMP-4-induced inhibition of myogenesis was lost by Smad4 ablation and restored by Smad4 overexpression. A nuclear zinc finger protein, E4F1, was identified as a possible component of the Smad4 complex that suppresses myogenic differentiation in response to BMP signaling. In the presence of Smad4, E4F1 stimulated the expression of Ids. Taken together, these findings suggest that the Smad signaling pathway may play a dual role in the BMP-induced conversion of myoblasts to osteoblastic cells.

Cardiotrophin-1 Maintains the Undifferentiated State in Skeletal Myoblasts

Skeletal myogenesis is potently regulated by the extracellular milieu of growth factors and cytokines. We observed that cardiotrophin-1 (CT-1), a member of the interleukin-6 (IL-6) family of cytokines, is a potent regulator of skeletal muscle differentiation. The normal up-regulation of myogenic marker genes, myosin heavy chain (MyHC), myogenic regulatory factors (MRFs), and myocyte enhancer factor 2s (MEF2s) were inhibited by CT-1 treatment. CT-1 also represses myogenin (MyoG) promoter activation. CT-1 activated two signaling pathways: signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinase kinase (MEK), a component of the extracellular signal-regulated MAPK (ERK) pathway. In view of the known connection between CT-1 and STAT3 activation, we surprisingly found that pharmacological blockade of STAT3 activity had no effect on the inhibition of myogenesis by CT-1 suggesting that STAT3 signaling is dispensable for myogenic repression. Conversely, MEK inhibition potently reversed the inhibition of myotube formation and attenuated the repression of MRF transcriptional activity mediated by CT-1. Taken together, these data indicate that CT-1 represses skeletal myogenesis through interference with MRF activity by activation of MEK/ERK signaling. In agreement with these in vitro observations, exogenous systemic expression of CT-1 mediated by adenoviral vector delivery increased the number of myonuclei in normal post-natal mouse skeletal muscle and also delayed skeletal muscle regeneration induced by cardiotoxin injection. The expression pattern of CT-1 in embryonic and post-natal skeletal muscle and in vivo effects of CT-1 on myogenesis implicate CT-1 in the maintenance of the undifferentiated state in muscle progenitor cells.

Inhibition of myogenesis by Notch: evidence for multiple pathways.

Notch signaling is critical for skeletal muscle development and regeneration, permitting the expansion of progenitor cells by preventing premature differentiation. We have interrogated the pathways through which ligand-mediated signaling inhibits myogenesis by identifying Notch target genes and assessing their impact on differentiation in vitro. Notch activation led to the robust induction of the transcriptional repressors Hey1 and HeyL in myoblasts, but only constitutive expression of Hey1 blocked myogenesis. siRNA-mediated knockdown of Hey1 had no effect on Notch's ability to inhibit differentiation, suggesting the existence of additional, possibly redundant pathways. We identified 82 genes whose expression was activated when C2C12 myoblasts were cultured in the presence of the Notch ligand Dll4. One of these, MyoR, is a novel Notch-responsive gene, whose protein product is known to repress myogenesis in vitro. siRNA-mediated knockdown of MyoR alone, or in combination with Hey1, was also ineffective at rescuing differentiation in the presence of Dll4. Our data support a model in which Notch signaling inhibits myogenesis through multiple pathways, two of which are defined by the Notch target genes Hey1 and MyoR.

Small-Molecule Fluorophores To Detect Cell-State Switching in the Context of High-Throughput Screening

A small molecule capable of distinguishing the distinct states resulting from cellular differentiation would be of enormous value, for example, in efforts aimed at regenerative medicine. We screened a collection of fluorescent small molecules for the ability to distinguish the differentiated state of a mouse skeletal muscle cell line. High-throughput fluorescence-based screening of C2C12 myoblasts and myotubes resulted in the identification of six compounds with the desired selectivity, which was confirmed by high-content screening in the same cell states. The compound that resulted in the greatest fluorescence intensity difference between the cell states was used as the screening agent in a pilot screen of 84 kinase inhibitors, each present in four doses, for inhibition of myogenesis. Of the kinase inhibitors, 17 resulted in reduction of fluorescence at one or more concentrations; among the "hits" included known inhibitors of myogenesis, confirming that this compound is capable of detecting the differentiated myotube state. We suggest that the strategy of screening for screening agents reported here may be extended more broadly in the future.

Transforming growth factor- -regulated miR-24 promotes skeletal muscle differentiation

MicroRNAs (miRNAs) have recently been proposed as a versatile class of molecules involved in regulation of a variety of biological processes. However, the role of miRNAs in TGF-β-regulated biological processes is poorly addressed. In this study, we found that miR-24 was upregulated during myoblast differentiation and could be inhibited by TGF-β1. Using both a reporter assay and Northern blot analysis, we showed that TGF-β1 repressed miR-24 transcription which was dependent on the presence of Smad3 and a Smads binding site in the promoter region of miR-24. TGF-β1 was unable to inhibit miR-24 expression in Smad3-deficient myoblasts, which exhibited accelerated myogenesis. Knockdown of miR-24 led to reduced expression of myogenic differentiation markers in C2C12 cells, while ectopic expression of miR-24 enhanced differentiation, and partially rescued inhibited myogenesis by TGF-β1. This is the first study demonstrating a critical role for miRNAs in modulating TGF-β-dependent inhibition of myogenesis, and provides a novel mechanism of the genetic regulation of TGF-β signaling during skeletal muscle differentiation.

Ectopic expression of porcine peroxisome proliferator-activated receptor δ regulates adipogenesis in mouse myoblasts1

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a critical role in regulating adipogenesis. The expression of peroxisome proliferator-activated receptor delta (PPARdelta) precedes that of PPARgamma during adipocyte differentiation in rodents. The current experiment was designed to study the function of porcine PPARdelta and the interaction of PPARdelta and PPARgamma in adipocyte differentiation. Inhibition of myogenesis was observed in mouse myoblasts expressing porcine PPARdelta, similar to myoblasts expressing PPARgamma. Treatment of myoblasts expressing PPARdelta with ligands for both PPARdelta and PPARgamma enhanced lipogenesis and adipogenesis to a greater extent than treatment with a PPARgamma ligand alone, suggesting that both genes were involved in regulating lipogenesis and adipogenesis. The ability to transdifferentiate myoblasts into adipocytes was decreased in myoblasts coexpressing PPARdelta with either wild type or mutated PPARgamma (Ser 112 was mutated to Ala; the mutated PPARgamma is more active than the wild type) compared with myoblasts expressing PPARgamma alone. Adipocyte differentiation in myoblasts coexpressing PPARdelta and mutated PPARgamma was greater than in myoblasts coexpressing PPARdelta and wild type PPARgamma, confirming that Ser 112 is important for the function of PPARgamma. Taken together, our results demonstrate that overexpression of PPARdelta inhibits myotube formation and also enhances adipocyte differentiation. However, the complexity and interaction of PPARdelta and PPARgamma in adipogenesis are not clearly understood.

Expression pattern of muscleblind-like proteins differs in differentiating myoblasts

Muscleblind-like (MBNL) proteins are believed to be regulators of myogenesis and are implicated in myotonic dystrophy. While Drosophila melanogaster muscleblind is required for terminal muscle differentiation, mammalian MBNL3 functions as an inhibitor of myogenesis. In this study, we analyzed the expression pattern of MBNL3 in different adult mouse tissues and tissue culture cells. MBNL3 transcript is enriched in the lung, spleen, and testis and not in heart and skeletal muscle. By Western blotting, we found that MBNL3 was expressed in C2C12 myoblasts and ts13 myofibroblasts, but was detected at significantly lower levels in fibroblasts. MBNL3 protein levels decreased when cells were shifted to muscle differentiation conditions, but the closely related MBNL1 protein was unaffected. These results suggest that myoblasts and fibroblasts respond to differentiation conditions by activating signaling pathways that repress MBNL3 but not MBNL1 expression.

Ran binding protein 9 interacts with Raf kinase but does not contribute to downstream ERK1/2 activation in skeletal myoblasts

Raf kinase is the upstream activator of MEK1/2 leading to phosphorylation and activation of ERK1/2. Sustained activation of Raf represses skeletal muscle-specific reporter gene transcription and formation of multinucleated myofibers. Inhibition of myogenesis by activated Raf involves downstream ERK1/2 as well as undefined mediators. To identify Raf-interacting proteins that may influence repression of muscle formation, a yeast two-hybrid screen was performed using a MEK1-binding defective Raf (RafBXB-T481A) as bait. Twenty cDNAs coding for Raf-interacting proteins were identified including Ran binding protein 9 (RanBP9), a protein previously reported to interact with receptor tyrosine kinases. Forced expression of RanBP9 in myogenic cells did not alter myogenesis. Co-expression of RanBP9 with constitutively active RafBXB, but not RafBXB-T481A, synergistically inhibited MyoD-directed muscle reporter gene transcription. Knockdown of RanBP9 expression did not restore the differentiation program to Raf-expressing myoblasts. Thus, RanBP9 physically associates with Raf but does not substantially contribute to the inhibitory actions of the kinase.

Myostatin auto‐regulates its expression by feedback loop through Smad7 dependent mechanism

Myostatin, a secreted growth factor, is a member of the TGF-beta superfamily and an inhibitor of myogenesis. Previously, we have shown that myostatin gene expression is regulated at the level of transcription and that myostatin is a downstream target gene of MyoD. Here we show that myostatin gene expression is auto-regulated by a negative feedback mechanism. Northern blot analysis indicated that there are relatively higher levels of myostatin mRNA in the biceps femoris muscle of cattle that express a non- functional myostatin allele (Belgian Blue) as compared to normal cattle. In contrast, addition of exogenous myostatin decreases endogenous myostatin mRNA. Consistent with this result, wild type myostatin protein is able to repress myostatin promoter activity via Activin type IIb receptor (ActRIIB) and ALK5 (P < 0.001). However, non-functional myostatin (Piedmontese) failed to repress the myostatin promoter suggesting that myostatin auto-regulates its promoter by negative feedback inhibition. Auto-regulation by myostatin appears to be signaled through Smad7, since the expression of the inhibitory Smad7 is induced by myostatin and the over-expression of Smad7 in turn inhibits the myostatin promoter activity (P < 0.001). In contrast down regulation of Smad7 by siRNA results in increased myostatin mRNA indicating that Smad7 is a negative regulator of myostatin gene expression. Consistent with these results, a decrease in Smad7 mRNA and concomitant increase in myostatin expression is seen in myotubes that express non functional myostatin. In addition, interference with myostatin signaling prevents the induction of Smad7 promoter activity by myostatin. Based on these results, we propose that myostatin auto-regulates its gene expression through a Smad7 dependent mechanism in myogenic cells.

Transforming Growth Factor β1 Is Up-regulated by Activated Raf in Skeletal Myoblasts but Does Not Contribute to the Differentiation-defective Phenotype

The Raf/MEK/MAPK signaling module elicits a strong negative impact on skeletal myogenesis that is reflected by a complete loss of muscle gene transcription and differentiation in multinucleated myocytes. Recent evidence indicates that Raf signaling also may contribute to myoblast cell cycle exit and cytoprotection. To further define the mechanisms by which Raf participates in cellular responses, a stable line of myoblasts expressing an estrogen receptor-Raf chimeric protein was created. The cells (23A2RafER(DD)) demonstrate a strict concentration-dependent increase in chimeric Raf protein synthesis and downstream phosphoMAPK activation. Initiation of low-level Raf activity in these cells augments contractile protein expression and myocyte fusion. By contrast, induction of high level Raf activity in 23A2RafER(DD) myoblasts inhibits the formation of myocytes and muscle reporter gene expression. Interestingly, treatment of myoblasts with conditioned medium isolated from Raf-repressive cells inhibits all of the aspects of myogenesis. Closer examination indicates that the transforming growth factor-beta(1) (TGF-beta(1)) gene is up-regulated in Raf-repressive myoblasts. The cells also direct elevated levels of Smad transcriptional activity, suggesting the existence of a TGF-beta(1) autocrine loop. However, extinguishing the biological activity of TGF-beta(1) does not restore the myogenic program. Our results provide evidence for the involvement of Raf signal transmission during myocyte formation as well as during inhibition of myogenesis.

Roles of peroxisome proliferator-activated receptors delta and gamma in myoblast transdifferentiation

Dietary long chain fatty acids and thiazolidinediones act as potent activators of adipogenesis in established preadipose cell lines. High concentrations of thiazolidinediones have also been shown to induce terminal differentiation of non-preadipose cells, such as fibroblasts and myoblasts, into adipose-like cells. This transdifferentiation was observed in both rodent and human myoblasts. In this report, we show that PPARδ mediates some of the effects exerted by long chain fatty acids on myogenesis and adipogenesis. Activation of PPARδ by long chain fatty acids impairs the expression of the determination factor MyoD1 and α-actin, abolishes the development of multinucleated myotubes, and in parallel induces the expression of PPARγ gene, a master regulator of adipogenesis. Ectopic expression of PPARδ in C2C12 myoblasts potentiated the fatty acid-induced expression of adipogenic markers, while expression of a dominant negative PPARδ mutant exerted opposite effects. Furthermore, a sequential activation of first PPARδ with long chain fatty acids and then PPARγ with thiazolidinediones is required for adipogenesis in C2C12 myoblasts. This study demonstrates that PPARδ, at least in part, is responsible for the dual effects of long chain fatty acids as inhibitors of myogenesis and inducers of transdifferentiation into preadipose-like cells.

Betaglycan Expression Is Transcriptionally Up-regulated during Skeletal Muscle Differentiation: CLONING OF MURINE BETAGLYCAN GENE PROMOTER AND ITS MODULATION BY MyoD, RETINOIC ACID, AND TRANSFORMING GROWTH FACTOR-β

Betaglycan is a membrane-anchored proteoglycan co-receptor that binds transforming growth factor beta (TGF-beta) via its core protein and basic fibroblast growth factor through its glycosaminoglycan chains. In this study we evaluated the expression of betaglycan during the C(2)C(12) skeletal muscle differentiation. Betaglycan expression, as determined by Northern and Western blot, was up-regulated during the conversion of myoblasts to myotubes. The mouse betaglycan gene promoter was cloned, and its sequence showed putative binding sites for SP1, Smad3, Smad4, muscle regulatory factor elements such as MyoD and MEF2, and retinoic acid receptor. Transcriptional activity of the mouse betaglycan promoter reporter was also up-regulated in differentiating C(2)C(12) cells. We found that MyoD, but not myogenin, stimulated this transcriptional activity even in the presence of high serum. Betaglycan promoter activity was increased by RA and inhibited by the three isoforms of TGF-beta. On the other hand, basic fibroblast growth factor, BMP-2, and hepatocyte growth factor/scatter factor, which are inhibitors of myogenesis, had little effect. In myotubes, up-regulated betaglycan was also detectable by TGF-beta affinity labeling and immunofluorescence microscopy studies. The latter indicated that betaglycan was localized both on the cell surface and in the ECM. Forced expression of betaglycan in C(2)C(12) myoblasts increases their responsiveness to TGF-beta2, suggesting that it performs a TGF-beta presentation function in this cell lineage. These results indicate that betaglycan expression is up-regulated during myogenesis and that MyoD and RA modulate its expression by a mechanism that is independent of myogenin.

Transforming growth factor β activates Rac1 and Cdc42Hs GTPases and the JNK pathway in skeletal muscle cells

The transforming growth factor β (TGFβ) plays an important role in cell growth and differentiation. However, the intracellular signaling pathways through which TGFβ inhibits skeletal myogenesis remain largely undefined. By measuring GTP-loading of Rho GTPases and the organization of the F-actin cytoskeleton and the plasma membrane, we analyzed the effect of TGFβ addition on the activity of three GTPases, Rac1, Cdc42Hs and RhoA. We report that TGFβ activates Rac1 and Cdc42Hs in skeletal muscle cells, two GTPases previously described to inhibit skeletal muscle cell differentiation whereas it inactivates RhoA, a positive regulator of myogenesis. We further show that TGFβ activates the C-jun N-terminal kinases (JNK) pathway in myoblastic cells through Rac1 and Cdc42Hs GTPases. We propose that the activation of Rho family proteins Rac1 and Cdc42Hs which subsequently regulate JNK activity participates in the inhibition of myogenesis by TGFβ.

Inhibition of Muscle Differentiation by the Novel Muscleblind-Related Protein CHCR

Growth factor withdrawal from proliferating myoblasts induces the expression of muscle-specific genes essential for myogenesis. By suppression subtractive hybridization (SSH), we have cloned a novel human cDNA that encodes a Cys3His zinc finger protein named CHCR (Cys3His CCG1-Required). CHCR is related to Muscleblind (Mbl), a Drosophila melanogaster protein required for terminal muscle differentiation. It also displays sequence similarity to EXP/MBNL, a human Mbl protein that interacts with CUG expansions associated with the degenerative muscular disease, myotonic dystrophy (DM1). This relationship with EXP/MBNL and Mbl suggests that CHCR also functions during muscle differentiation. We have found that CHCR mRNA and protein levels decrease upon differentiation of mouse myoblast cells. Constitutive expression of CHCR in C2C12 cells inhibits the induction of sarcomeric myosin heavy chain (MyHC) upon serum deprivation. Induction of myogenin, an earlier marker of muscle differentiation, is inhibited to a lesser extent, while expression of the cell cycle inhibitor, p21, remains unaffected. Loss of CHCR function by morpholino antisense oligonucleotide treatment accelerates MyHC induction during differentiation of myoblast cells. These complementary gain- and loss-of-function results suggest that CHCR is an inhibitor of myogenesis. CHCR represents the first muscleblind-related protein that antagonizes, instead of promotes, muscle differentiation.

CDNA microarray analysis of global gene expression in sarcomas.

Sequencing of the human genome and rapidly evolving microarray technology have combined to provide investigators with the ability to analyze individual tumors and groups of tumors for global patterns of gene expression. Few of these types of studies have been performed on rhabdomyosarcomas and osteogenic sarcomas, including cell lines and animal models. Groups of expressed genes that may characterize rhabdomyosarcomas and their subgroups and separate them from other types of tumors have been identified. More specifically, genes involved in myogenesis or the inhibition of myogenesis have been identified, as have genes that may play a role in metastatic activity in osteogenic sarcomas. Also, a study documenting the consistent and specific gene expression profile of gastrointestinal stromal tumors has been published. While the data regarding gene expression patterns in sarcomas is accruing, numerous investigators are working on developing and enhancing bioinformatic skills and tools such that the vast amount of data can be converted into knowledge regarding biology, therapeutic responsiveness or resistance, and prognosis.