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The f subunit of the eukaryotic initiation factor 3 (eIF3f) is downregulated in several cancers and in particular in melanoma and pancreatic cancer cells. Its enforced expression by transient gene transfection negatively regulates cancer cell growth by activating apoptosis. With the aim to increase the intracellular level of eIF3f proteins and activate apoptosis in cancer cell lines, we developed a protein transfer system composed of a cell-penetrating peptide sequence fused to eIF3f protein sequence (MD11-eIF3f). To determine whether exogenously administered eIF3f proteins were able to compensate the loss of endogenous eIF3f and induce cancer cell death, we analyzed the therapeutic action of MD11-eIF3f in several tumor cells. We identified four cell lines respondent to eIF3f-treatment and we evaluated the antitumor properties of the recombinant proteins using dose- and time-dependent studies. Our results demonstrate that this protein delivery approach represents an innovative and powerful strategy for cancer treatment.

The eukaryotic initiation factor 3 subunit f (eIF3f) is one of the 13 subunits of the translation initiation factor complex eIF3 required for several steps in the initiation of mRNA translation. In skeletal muscle, recent studies have demonstrated that eIF3f plays a central role in skeletal muscle size maintenance. Accordingly, eIF3f overexpression results in hypertrophy through modulation of protein synthesis via the mTORC1 pathway. Importantly, eIF3f was described as a target of the E3 ubiquitin ligase MAFbx/atrogin-1 for proteasome-mediated breakdown under atrophic conditions. The biological importance of the MAFbx/atrogin-1-dependent targeting of eFI3f is highlighted by the finding that expression of an eIF3f mutant insensitive to MAFbx/atrogin-1 polyubiquitination is associated with enhanced protection against starvation-induced muscle atrophy. A better understanding of the precise role of this subunit should lead to the development of new therapeutic approaches to prevent or limit muscle wasting that prevails in numerous physiological and pathological states such as immobilization, aging, denervated conditions, neuromuscular diseases, AIDS, cancer, diabetes. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The mTORC1 pathway is required for both the terminal muscle differentiation and hypertrophy by controlling the mammalian translational machinery via phosphorylation of S6K1 and 4E-BP1. mTOR and S6K1 are connected by interacting with the eIF3 initiation complex. The regulatory subunit eIF3f plays a major role in muscle hypertrophy and is a key target that accounts for MAFbx function during atrophy. Here we present evidence that in MAFbx-induced atrophy the degradation of eIF3f suppresses S6K1 activation by mTOR, whereas an eIF3f mutant insensitive to MAFbx polyubiquitination maintained persistent phosphorylation of S6K1 and rpS6. During terminal muscle differentiation a conserved TOS motif in eIF3f connects mTOR/raptor complex, which phosphorylates S6K1 and regulates downstream effectors of mTOR and Cap-dependent translation initiation. Thus eIF3f plays a major role for proper activity of mTORC1 to regulate skeletal muscle size.

We recently presented evidence that the subunit eIF3-f of the eukaryotic initiation translation factor eIF3 that interacts with the E3-ligase Atrogin-1/muscle atrophy F-box (MAFbx) for polyubiquitination and proteasome-mediated degradation is a key target that accounts for MAFbx function during muscle atrophy. To understand this process, deletion analysis was used to identify the region of eIF3-f that is required for its proteolysis. Here, we report that the highly conserved C-terminal domain of eIF3-f is implicated for MAFbx-directed polyubiquitination and proteasomal degradation. Site-directed mutagenesis of eIF3-f revealed that the six lysine residues within this domain are required for full polyubiquitination and degradation by the proteasome. In addition, mutation of these six lysines (mutant K(5-10)R) displayed hypertrophic activity in cellulo and in vivo and was able to protect against starvation-induced muscle atrophy. Taken together, our data demonstrate that the C-terminal modifications, believed to be critical for proper eIF3-f regulation, are essential and contribute to a fine-tuning mechanism that plays an important role for eIF3-f function in skeletal muscle.

Ubiquitin ligase Atrogin1/Muscle Atrophy F-box (MAFbx) up-regulation is required for skeletal muscle atrophy but substrates and function during the atrophic process are poorly known. The transcription factor MyoD controls myogenic stem cell function and differentiation, and seems necessary to maintain the differentiated phenotype of adult fast skeletal muscle fibres. We previously showed that MAFbx mediates MyoD proteolysis in vitro. Here we present evidence that MAFbx targets MyoD for degradation in several models of skeletal muscle atrophy. In cultured myotubes undergoing atrophy, MAFbx expression increases, leading to a cytoplasmic-nuclear shuttling of MAFbx and a selective suppression of MyoD. Conversely, transfection of myotubes with sh-RNA-mediated MAFbx gene silencing (shRNAi) inhibited MyoD proteolysis linked to atrophy. Furthermore, overexpression of a mutant MyoDK133R lacking MAFbx-mediated ubiquitination prevents atrophy of mouse primary myotubes and skeletal muscle fibres in vivo. Regarding the complex role of MyoD in adult skeletal muscle plasticity and homeostasis, its rapid suppression by MAFbx seems to be a major event leading to skeletal muscle wasting. Our results point out MyoD as the second MAFbx skeletal muscle target by which powerful therapies could be developed.

The control of muscle cell size is a physiological process balanced by a fine tuning between protein synthesis and protein degradation. MAFbx/Atrogin-1 is a muscle specific E3 ubiquitin ligase upregulated during disuse, immobilization and fasting or systemic diseases such as diabetes, cancer, AIDS and renal failure. This response is necessary to induce a rapid and functional atrophy. To date, the targets of MAFbx/Atrogin-1 in skeletal muscle remain to be identified. We have recently presented evidence that eIF3-f, a regulatory subunit of the eukaryotic translation factor eIF3 is a key target that accounts for MAFbx/Atrogin-1 function in muscle atrophy. More importantly, we showed that eIF3-f acts as a "translational enhancer" that increases the efficiency of the structural muscle proteins synthesis leading to both in vitro and in vivo muscle hypertrophy. We propose that eIF3-f subunit, a mTOR/S6K1 scaffolding protein in the IGF-1/Akt/mTOR dependent control of protein translation, is a positive actor essential to the translation of specific mRNAs probably implicated in muscle hypertrophy. The central role of eIF3-f in both the atrophic and hypertrophic pathways will be discussed in the light of its promising potential in muscle wasting therapy.

In response to cancer, AIDS, sepsis and other systemic diseases inducing muscle atrophy, the E3 ubiquitin ligase Atrogin1/MAFbx (MAFbx) is dramatically upregulated and this response is necessary for rapid atrophy. However, the precise function of MAFbx in muscle wasting has been questioned. Here, we present evidence that during muscle atrophy MAFbx targets the eukaryotic initiation factor 3 subunit 5 (eIF3-f) for ubiquitination and degradation by the proteasome. Ectopic expression of MAFbx in myotubes induces atrophy and degradation of eIF3-f. Conversely, blockade of MAFbx expression by small hairpin RNA interference prevents eIF3-f degradation in myotubes undergoing atrophy. Furthermore, genetic activation of eIF3-f is sufficient to cause hypertrophy and to block atrophy in myotubes, whereas genetic blockade of eIF3-f expression induces atrophy in myotubes. Finally, eIF3-f induces increasing expression of muscle structural proteins and hypertrophy in both myotubes and mouse skeletal muscle. We conclude that eIF3-f is a key target that accounts for MAFbx function during muscle atrophy and has a major role in skeletal muscle hypertrophy. Thus, eIF3-f seems to be an attractive therapeutic target.

The transcription factors MyoD and Myf5 present distinct patterns of expression during cell cycle progression and development. In contrast to the mitosis-specific disappearance of Myf5, which requires a D-box-like motif overlapping the basic domain, here we describe a stable and inactive mitotic form of MyoD phosphorylated on its serine 5 and serine 200 residues by cyclin B-cdc2. In mitosis, these modifications are required for releasing MyoD from condensed chromosomes and inhibiting its DNA-binding and transcriptional activation ability. Then, nuclear MyoD regains instability in the beginning of G1 phase due to rapid dephosphorylation events. Moreover, a non-phosphorylable MyoD S5A/S200A is not excluded from condensed chromatin and alters mitotic progression with apparent abnormalities. Thus, the drop of MyoD below a threshold level and its displacement from the mitotic chromatin could present another window in the cell cycle for resetting the myogenic transcriptional program and to maintain the myogenic determination of the proliferating cells.

MyoD controls myoblast identity and differentiation and is required for myogenic stem cell function in adult skeletal muscle. MyoD is degraded by the ubiquitin-proteasome pathway mediated by different E3 ubiquitin ligases not identified as yet. Here we report that MyoD interacts with Atrogin-1/MAFbx (MAFbx), a striated muscle-specific E3 ubiquitin ligase dramatically up-regulated in atrophying muscle. A core LXXLL motif sequence in MyoD is necessary for binding to MAFbx. MAFbx associates with MyoD through an inverted LXXLL motif located in a series of helical leucine-charged residue-rich domains. Mutation in the LXXLL core motif represses ubiquitination and degradation of MyoD induced by MAFbx. Overexpression of MAFbx suppresses MyoD-induced differentiation and inhibits myotube formation. Finally the purified recombinant SCF(MAFbx) complex (SCF, Skp1, Cdc53/Cullin 1, F-box protein) mediated MyoD ubiquitination in vitro in a lysine-dependent pathway. Mutation of the lysine 133 in MyoD prevented its ubiquitination by the recombinant SCF(MAFbx) complex. These observations thus demonstrated that MAFbx functions in ubiquitinating MyoD via a sequence found in transcriptional coactivators. These transcriptional coactivators mediate the binding to liganded nuclear receptors. We also identified a novel protein-protein interaction module not yet identified in F-box proteins. MAFbx may play an important role in the course of muscle differentiation by determining the abundance of MyoD.

The ubiquitin-proteasome system is responsible for the regulation and turnover of the nuclear transcription factor MyoD. The degradation of MyoD can occur via an NH2 terminus-dependent pathway or a lysine-dependent pathway, suggesting that MyoD ubiquitination may be driven by different mechanisms. To understand this process, deletion analysis was used to identify the region of MyoD that is required for rapid proteolysis in the lysine-dependent pathway. Here we report that the basic helix-loop-helix domain is required for ubiquitination and lysine-dependent degradation of MyoD in the nucleus. Site-directed mutagenesis in MyoD revealed that lysine 133 is the major internal lysine of ubiquitination. The half-life of the MyoD K133R mutant protein was longer than that of wild type MyoD, substantiating the implication of lysine 133 in the turnover of MyoD in myoblasts. In addition, the MyoD K133R mutant displayed activity 2-3-fold higher than the wild type in transactivation muscle-specific gene and myogenic conversion of 10T1/2 cells. Taken together, our data demonstrate that lysine 133 is targeted for ubiquitination and rapid degradation of MyoD in the lysine-dependent pathway and plays an integral role in compromising MyoD activity in the nucleus.

The transcription factors MyoD and Myf-5 control myoblast identity and differentiation. MyoD and Myf-5 manifest opposite cell cycle-specific expression patterns. Here, we provide evidence that MyoD plays a pivotal role at the G(2)/M transition by controlling the expression of p21(Waf1/Cip1) (p21), which is believed to regulate cyclin B-Cdc2 kinase activity in G(2). In growing myoblasts, MyoD reaccumulates during G(2) concomitantly with p21 before entry into mitosis; MyoD is phosphorylated on Ser5 and Ser200 by cyclin B-Cdc2, resulting in a decrease of its stability and down-regulation of both MyoD and p21. Inducible expression of a nonphosphorylable MyoD A5/A200 enhances the MyoD interaction with the coactivator P/CAF, thereby stimulating the transcriptional activation of a luciferase reporter gene placed under the control of the p21 promoter. MyoD A5/A200 causes sustained p21 expression, which inhibits cyclin B-Cdc2 kinase activity in G(2) and delays M-phase entry. This G(2) arrest is not observed in p21(-/-) cells. These results show that in cycling cells MyoD functions as a transcriptional activator of p21 and that MyoD phosphorylation is required for G(2)/M transition.

Acute myeloid leukemia (AML) is sustained by the extensive proliferation of leukemic stem and progenitor cells, which give rise to the population of leukemic blasts with defective differentiation and low proliferative capacity. We have recently shown that ligation of CD44, a cell surface molecule present on AML cells, with specific monoclonal antibodies (mAbs) inhibits their proliferation. However, its mechanism has not been investigated yet. Here, using the NB4 cell line as a model of proliferating human AML cells, and the A3D8 mAb to ligate CD44, we show for the first time that CD44 ligation stabilizes the cyclin-dependent kinase inhibitor p27(Kip1) (p27) protein, resulting in increased association with cyclin E/Cdk2 complexes and inhibition of their kinase activity. Moreover, using a p27 antisense vector, we provide direct evidence that p27 is the main mediator of cell growth arrest by CD44. CD44 ligation also leads to p27 accumulation in THP-1, KG1a, and HL60 cell lines and in primary leukemic cells, suggesting that this process is general in AML. Taken together, our present results suggest that CD44 is a new and efficient means to increase the expression of p27 in AML cells. Considering that elevated expression of p27 is a factor of good prognosis in AML, these results provide a new basis for developing CD44-targeted therapy in AML.

Rhabdomyosarcoma (RMS) has deregulated proliferation and is blocked in the differentiation program despite Myf-5, MyoD and myogenin expression. Here we show that ectopic expression of MRF4, which is not subject to an autoregulatory pathway but regulated by the other MRFs protein family, induces growth arrest and terminal differentiation in RD cells. Deletion mapping identified a positive-acting C-terminal domain in MRF4 as the mediator of transcriptional activity, revealing a conserved motif with helix III in MyoD previously found to initiate expression of endogenous skeletal muscle genes. By using chimeric MyoD/MRF4 proteins, we observe that the C-terminal motif of MRF4 rescues MyoD activity in RD cells. Moreover, comparative induction of muscle-specific genes following activation of MyoD, through the expression of a constitutively activated MKK6 either in the absence or presence of MRF4, shows that MyoD and MRF4 can differently regulate muscle genes expression. Together, these results demonstrate that the MRF4 C-terminus functions as specification as well as activation domain in tumor cells. They provide a basis to identify gene products necessary for b-HLH-mediated differentiation versus tumor progression.

The cyclin-dependent kinase inhibitor p57(Kip2) is required for normal mouse embryonic development. p57(Kip2) consists of four structurally distinct domains in which the conserved C-terminal nuclear targeting domain contains a putative Cdk phosphorylation site (Thr(342)) that shares a great similitude in the adjacent sequences with p27(Kip1) but not with p21(Cip1). Phosphorylation on Thr(187) has been shown to promote degradation of p27(Kip1). Although there is sequence homology between the C-terminal part of p27(Kip1) and p57(Kip2), we show that the ubiquitination and degradation of p57(Kip2) are independent of Thr(342). In contrast a destabilizing element located in the N-terminal is implicated in p57(Kip2) destabilization.

The transcription factor MyoD, member of the myogenic regulators family, induces differentiation in precursor cells by its ability to arrest cell proliferation and to activate muscle specific genes. MyoD plays a key role in the antagonism between proliferation and differentiation. The withdrawal from the cell cycle and the activation of muscle differentiation are related to the level of MyoD protein. The cyclin E-cdk2 complex, one of the key regulators of the G1/S transition is directly implicated in the degradation of MyoD by the ubiquitin-proteasome pathway, leading the myoblasts to proliferate. The display of this control in normal myoblasts suggests that its deficiency in the muscle stem cells could lead to the formation of rhabdomyosarcomas which have lost both the control of cell proliferation and the transcriptional activity of MyoD.

Proliferating myoblasts already express MyoD before the induction of differentiation. Overexpression of MyoD in normal and transformed cell lines was shown to block cells from entering S phase, suggesting that the MyoD growth suppressive effect must be tightly controlled in growing myoblasts. Here we show that during G1 phase, but not in G2, MyoD abundance is down-regulated by the ubiquitin-proteasome pathway through phosphorylation of serine 200. Roscovitine, a specific inhibitor of cyclin-Cdk2 complexes, prevents both phosphorylation and degradation of MyoD in G1. Inhibition of the ubiquitin-dependent proteasome pathway by MG132 results in stabilization of MyoD-wt, with little effect on a MyoD mutant where serine 200 is replaced by an alanine. Our results show that MyoD Ser200 is the substrate for phosphorylation by cyclin E-Cdk2 stimulating its degradation by the ubiquitin-proteasome system which controls MyoD levels in G1. Phosphorylation/degradation of MyoD at the end of G1 thus represents the regulatory checkpoint in growing myoblasts allowing progression into S phase in a manner similar to the recently examplified cdk2-phosphorylation/degradation of p27(Kip1)., (Copyright 2000 Academic Press.)

Recent data have demonstrated the role of Cdk1- and Cdk2-dependent phosphorylation of MyoD(Ser200) in the regulation of MyoD activity and protein turnover. In the present study, we show that in presence of p57(Kip2), MyoD(Ala200), a MyoD mutant that cannot be phosphorylated by cyclin-Cdk complexes, displayed activity 2-5-fold higher than of MyoD(Ala200) alone in transactivation of muscle-specific genes myosin heavy chain, creatine kinase, and myosin light chain 1. Furthermore, p57(Kip2) increases the levels of MyoD(Ala200) in cotransfected cells. This result implies that p57(Kip2) may regulate MyoD through a process distinct from its function as a cyclin-dependent kinase inhibitors. We report that overexpression of p57(Kip2) increased the half-life of MyoD(Ala200). This increased half-life of MyoD involves a physical interaction between MyoD and p57(Kip2) but not with p16(Ink4a), as shown by cross-immunoprecipitation not only on overexpressed proteins from transfected cells, but also on endogenous MyoD and p57(Kip2) from C2C12 myogenic cells. Mutational and functional analyses of the two proteins show that the NH(2) domain of p57(Kip2) associates with basic region in the basic helix-loop-helix domain of MyoD. Competition/association assays and site-directed mutagenesis of the NH(2) terminus of p57(Kip2) identified the intermediate alpha-helix domain, located between the Cdk and the cyclin binding sites, as essential for MyoD interaction. These data show that the alpha-helix domain of p57(Kip2), which is conserved in the Cip/Kip proteins, is implicated in protein-protein interaction and confers a specific regulatory mechanism, outside of their Cdk-inhibitory activity, by which the p57(Kip2) family members positively act on myogenic differentiation.

Previous studies have led to the proposal that a single molecule of Cki can associate with the cyclin/Cdk complex to repress its activity. On the other hand, multiple inhibitor molecules are required to inhibit Cdks. In the present work, by using differently tagged p57Kip2 proteins we demonstrate that p57Kip2 can bind to itself in vitro and in vivo. Mutational deletion analysis showed that the NH2 terminal domain of p57Kip2 is necessary and sufficient to dimerization. Using an in vitro competition/association assay, we demonstrate that cyclin D1 alone, Cdk4 alone and/or cyclin D1/Cdk4 complexes do not compete for the p57Kip2 homodimers formation. However, a mutation in the alpha-helix domain of p57Kip2 (R33L) strongly reduced homodimer formation but did not modify interaction with cyclin D1-Cdk4 complexes. Also, increasing amounts of p57Kip2 lead in vivo to a significant augmentation in the level of p57Kip2 homodimerization associated with cyclin D1-Cdk4 complexes and to a marked inhibition of the cyclin D1-Cdk4 kinase activity. Altogether, these data suggest a model whereby p57Kip2 associates with itself by using the NH2 domain to form a homodimeric species which interacts with and inhibits the cyclin D1-Cdk4 complexes.

When ectopically expressed, the serine/threonine kinase Mos can induce oncogenic transformation of somatic cells by direct phosphorylation of MAP kinase/ERK kinase (MEK1), activating the mitogen-activated protein kinases ERK1 and ERK2. On the other hand, overexpression of Mos in C2C12 myoblasts is not transforming. Mos activates myogenic differentiation by promoting heterodimerization of the MyoD/E12 proteins, increasing the expression of myogenic markers and the positive autoregulatory loop of MyoD. In this study, we show that in myogenic cells, the mitogenic and oncogenic signalling from the Mos/MEK/ERK pathway is suppressed by MyoD through the formation of a heterotrimeric complex.

We show that expression of p57(Kip2), a potent tight-binding inhibitor of several G(1) cyclin-cyclin-dependent kinase (Cdk) complexes, increases markedly during C2C12 myoblast differentiation. We examined the effect of p57(Kip2) on the activity of the transcription factor MyoD. In transient transfection assays, transcriptional transactivation of the mouse muscle creatine kinase promoter by MyoD was enhanced by the Cdk inhibitors. In addition, p57(Kip2), p21(Cip1), and p27(Kip1) but not p16(Ink4a) induced an increased level of MyoD protein, and we show that MyoD, an unstable nuclear protein, was stabilized by p57(Kip2). Forced expression of p57(Kip2) correlated with hypophosphorylation of MyoD in C2C12 myoblasts. A dominant-negative Cdk2 mutant arrested cells at the G(1) phase transition and induced hypophosphorylation of MyoD. Furthermore, phosphorylation of MyoD by purified cyclin E-Cdk2 complexes was inhibited by p57(Kip2). In addition, the NH2 domain of p57(Kip2) necessary for inhibition of cyclin E-Cdk2 activity was sufficient to inhibit MyoD phosphorylation and to stabilize it, leading to its accumulation in proliferative myoblasts. Taken together, our data suggest that repression of cyclin E-Cdk2-mediated phosphorylation of MyoD by p57(Kip2) could play an important role in the accumulation of MyoD at the onset of myoblast differentiation.

Urokinase can form a tripartite complex binding urokinase receptor (uPAR) and plasminogen activator inhibitor type-1 (PAI-1), a component of the extracellular matrix (ECM). The components of the tripartite complex are modulated throughout the in vitro myogenic differentiation process. A series of experiments aimed at elucidating the role of the urokinase tripartite complex in the fusion of human myogenic cells were performed in vitro. Myogenic cell fusion was associated with increased cell-associated urokinase-type plasminogen activator (uPA) activity, cell-associated uPAR, and uPAR occupancy. Incubation of cultures with either uPA anticatalytic antibodies, or the amino-terminal fragment of uPA (ATF), which inhibits competitively uPA binding to its receptor, or anti-PAI-1 antibodies, which inhibit uPA binding to PAI-1, resulted in a 30 to 47% decrease in fusion. Incubation of cultures with the plasmin inhibitor aprotinin did not affect fusion. Decreased fusion rates induced by interfering with uPAR/uPA/PAI-1 interactions were not associated with significant changes in mRNA levels of both the myogenic regulatory factor myogenin and its inhibitor of DNA binding, Id. Incubation of cultures with purified uPA resulted in a decrease in fusion, likely due to a competitive inhibition of PAI-1 binding of endogenous uPA. We conclude that muscle cell fusion largely depends on interactions between the members of the urokinase complex (uPAR/uPA/PAI-1), but does not require proteolytic activation of plasmin. Since the intrinsic muscle cell differentiation program appears poorly affected by the state of integrity of the urokinase complex, and since cell migration is a prerequisite for muscle cell fusion in vitro, it is likely that the urokinase system is instrumental in fusion through its connection with the cell migration process. Our results suggest that the urokinase tripartite complex may be involved in cell migration in a non conventional way, playing the role of an adhesion system bridging cell membrane to ECM.

The activities of myogenic basic helix-loop-helix (bHLH) factors are regulated by a number of different positive and negative signals. Extensive information has been published about the molecular mechanisms that interfere with the process of myogenic differentiation, but little is known about the positive signals. We previously showed that overexpression of rat Mos in C2C12 myoblasts increased the expression of myogenic markers whereas repression of Mos products by antisense RNAs inhibited myogenic differentiation. In the present work, our results show that the rat mos proto-oncogene activates transcriptional activity of MyoD protein. In transient transfection assays, Mos promotes transcriptional transactivation by MyoD of the muscle creatine kinase enhancer and/or a reporter gene linked to MyoD-DNA binding sites. Physical interaction between Mos and MyoD, but not with E12, is demonstrated in vivo by using the two-hybrid approach with C3H10T1/2 cells and in vitro by using the glutathione S-transferase (GST) pull-down assays. Unphosphorylated MyoD from myogenic cell lysates and/or bacterially expressed MyoD physically interacts with Mos. This interaction occurs via the helix 2 region of MyoD and a highly conserved region in Mos proteins with 40% similarity to the helix 2 domain of the E-protein class of bHLH factors. Phosphorylation of MyoD by activated GST-Mos protein inhibits the DNA-binding activity of MyoD homodimers and promotes MyoD-E12 heterodimer formation. These data support a novel function for Mos as a mediator (coregulator) of muscle-specific gene(s) expression.

We analysed the signaling pathways involved in myogenic differentiation of primary cultures of rat satellite cells using substances targeting the protein kinase C (PKC) and the cAMP protein kinase (PKA) pathways. We have previously shown that iso-H7, which putatively inhibits both PKC and PKA, strongly stimulates satellite cell differentiation, as well as the PKA inhibitor HA1004. In the study reported here, the effects of iso-H7 on satellite cell differentiation were compared to those observed in the presence of agents which reduce PKC activity. It was shown that treatments with the highly specific PKC inhibitor GF109203X or with 12-O-tetradecanoylphorbol 13-acetate (TPA) which induced a partial PKC downregulation, did not significantly alter myogenic differentiation. Northern blot analyses showed that iso-H7 activated the expression of myogenin but not that of MyoD mRNA. Concurrently, iso-H7 increased myosin light-chain mRNA expression. In contrast, TPA had no effect on these syntheses. Taken together, these results showed that iso-H7 did not act intracellularly as a PKC inhibitor but rather as a PKA inhibitor as previously suggested. Our results are compatible with the hypothesis that a reduction in PKA activity controls satellite cell myogenesis through an increased myogenin mRNA expression.

Terminal differentiation of muscle cells results in opposite effects on gene promoters: muscle-specific promoters, which are repressed during active proliferation of myoblasts, are turned on, whereas at least some proliferation-associated promoters, such as c-fos, which are active during cell division, are turned off. MyoD and myogenin, transcription factors from the basic-helix-loop-helix (bHLH) family, are involved in both processes, up-regulating muscle genes and down-regulating c-fos. On the other hand, the serum response factor (SRF) is involved in the activation of muscle-specific genes, such as c-fos, as well as in the up-regulation of a subset of genes that are responsive to mitogens. Upon terminal differentiation, the activity of these various transcription factors could be modulated by the formation of distinct protein-protein complexes. Here, we have investigated the hypothesis that the function of SRF and/or MyoD and myogenin could be modulated by a physical association between these transcription factors. We show that myogenin from differentiating myoblasts specifically binds to SRF. In vitro analysis, using the glutathione S-transferase pull-down assay, indicates that SRF-myogenin interactions occur only with myogenin-E12 heterodimers and not with isolated myogenin. A physical interaction between myogenin, E12, and SRF could also be demonstrated in vivo using a triple-hybrid approach in yeast. Glutathione S-transferase pull-down analysis of various mutants of the proteins demonstrated that the bHLH domain of myogenin and that of E12 were necessary and sufficient for the interaction to be observed. Specific binding to SRF was also seen with MyoD. In contrast, Id, a natural inhibitor of myogenic bHLH proteins, did not bind SRF in any of the situations tested. These data suggest that SRF, on one hand, and myogenic bHLH, on the other, could modulate each other's activity through the formation of a heterotrimeric complex.

Background: Some genetic alterations have been shown to have prognostic implication for patients with neuroblastoma: MYCN oncogene amplification, deletion of the short arm of chromosome 1 and di- or tetraploidy. The goal of this study was to analyze these factors in children with neuroblastoma., Methods: Twenty neuroblastoma samples were analyzed with morphologic cytogenetics, and each of them was compared with MYCN amplification status by Southern blot and fluorescent in situ hybridization (FISH) with a genomic probe., Results: A complete karyotype was obtained for 14 children. A diploid or tetraploid mode and a 1p deletion were found in most children with advanced stages. MYCN amplification status was totally concordant with both methods in all patients, even in a case with low level amplification. A wide intercellular variation in the amplification level in each MYCN amplified sample was shown., Conclusion: The use of FISH to assess MYCN amplification rapidly in neuroblastoma is recommended. This method could be very useful in future therapeutic protocols in which treatment is based on MYCN status (and especially for infants and children with localized tumor).

We have cloned and sequenced an almost complete c-DNA and the entire genomic sequence of rat the H19 gene, which is developmentally regulated in skeletal muscle. The data base comparison revealed a 92% homology with mouse gene H19. However the rat H19 ORFs do not display significant homology with the H19 ORFs from other species. In contrast to the mouse, the rat H19 mRNA is not easily detectable in fetal rat skeletal fibers. Its level increases after birth (up to 12 to 18 days) and remains stable thereafter. The pattern of H19 mRNA expression in rat muscle in vivo is very similar to the c-mos gene expression in this tissue, suggesting an interrelationship between H19 and c-mos products during muscle differentiation. Indeed, our results indicate that overexpression of c-mos protein in the muscle cell line C2C12 induces a concomitant increase of H19 mRNA expression. Furthermore, repression of c-mos protein expression by anti-sense RNAs extinguishes H19 mRNA expression and inhibits the differentiation process. These data suggest a relationship between c-mos and H19 expression and, in addition, the involvement of both gene products in the process of myogenesis.

Satellite cells are involved in physiological growth and post-traumatic regeneration of adult skeletal muscle fibres. In this study, it is shown that differentiation of primary cultures of rat satellite cells is increased by inhibitors of ser/thr protein kinases such as iso-H7, which both inhibit cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) activities, and HA1004, a PKA inhibitor. These results, showing a preponderant effect of PKA inhibition on myogenesis in vitro, prompted the effects of iso-H7 on muscular regeneration in vivo to be tested. Preliminary results showed that regeneration of rat muscle EDL was improved by iso-H7 treatment.

We have used fractionation of subcellular components of the skeletal muscle followed by Western blot analyses to study the localization of the c-mos protein in adult rat muscle. We find that p43c-mos is predominantly located in the KCl supernatant fraction. We show that immunoprecipitates of p43c-mos phosphorylate in vitro two polypeptides of about 34 kDa and 80 kDa respectively. Muscle fractionation and immunodetection studies showed that the p34 protein associated with p43c-mos is the cdc2 protein. p43c-mos is coprecipitated with p34cdc2 when using either anti PSTAIR antibody, antibody directed against the conserved COOH terminal region of the p34cdc2 and by binding to beads that contain cross-linked p13suc1, a protein known to bind p34cdc2. Likewise p34cdc2 coprecipitated with p43c-mos when using anti mos antibody. However p43c-mos is not present in histone H1 kinase active p34cdc2 complex precipitated with anti p34cdc2 COOH-terminal peptide antibody. In adult muscle tissue tubulin is not complexed with p34cdc2 and p43c-mos as previously observed in c-mos and v-mos transformed cells. Gel filtration and crosslinking experiments show that a 170 kDa complex contains c-mos and p34cdc2 proteins. In addition during postnatal development of skeletal muscle we observe modifications in the migration pattern of p34cdc2 correlated with the accumulation of p43c-mos. Our findings raise the possibility of a p43c-mos-p34cdc2 complex could play a role in the differentiation process and maintenance of myotubes in Go.

The changes occurring in the hematopoietic extracellular matrix in an experimental myeloproliferative syndrome were explored by comparing the glycosaminoglycan (GAG) composition of normal mouse spleens and spleens infected with myeloproliferative sarcoma virus (MPSV). Large quantities of hyaluronate and of sulfated GAGs accumulated in the extracellular matrix of infected spleens, as shown by histoimmunoassay and alcian blue staining, respectively. The splenic GAGs were either labeled with 35S-sulfate injected in vivo or unlabeled. The spleens were fractionated to separate hematopoietic cells from the stromal component containing extracellular matrix material and fibroblasts, and the GAGs were extracted from each fraction. Specific degradative treatments and electrophoresis indicated that sulfated GAGs were mostly chondroitin sulfate and heparan sulfate. Three hours after in vivo injection of 35S-sulfate, the amount of 35S-GAGs was increased approximately fivefold per mg stromal proteins. The bulk of these 35S-GAGs (70%) was recovered in the stromal fraction. The higher amount of sulfated GAGs in leukemic spleen was due both to the presence of more producer cells (infected fibroblasts and hematopoietic cells) and to a stimulation of GAG synthesis per cell, as evidenced 35S-labeling in in vitro experiments. Chondroitin sulfate was the main sulfated GAG present in the culture medium of both hematopoietic and fibroblastic cells and in the pericellular material released by trypsin from fibroblastic cells. High amounts of chondroitin sulfate, which has a possible role in the detachment of hematopoietic cells from the stromal cells, may favour the release of hematopoietic cells from the spleen into the peripheral blood. Heparan sulfate was produced by fibroblastic cells and it was principally present in their pericellular material. Considering the capacity of heparan sulfate to retain cytokines, as demonstrated by others in vitro, large amounts of heparan sulfate may result in the retention of large amounts of the cytokines, which production is enhanced in the infected spleen. This phenomenon may contribute to promote the hematopoietic stem cell proliferation characteristic of the MPSV-induced myeloproliferative disease.

We have isolated and sequenced a cDNA (mrfms) encoding rat c-fms gene (CSF-1 receptor) from proliferating L6 alpha 1 myoblasts. The predicted amino acid sequence was highly identical with the c-fms protein found in monocytes and macrophages (98, 76 and 84% identity from mouse, cat and human c-fms proteins, respectively). The mechanisms responsible for the regulation of mrfms gene expression during myogenesis were examined. Mrfms products were observed during proliferation of L6 alpha 1 myoblasts and were downregulated during differentiation. Run-on transcription assays demonstrated that the mrfms gene was transcriptionally active only in undifferentiated myoblasts. These findings suggested that mrfms levels in L6 alpha 1 myoblasts are controlled by transcriptional mechanisms. The half-life of mrfms transcripts was found to be at least 5 hr while inhibition of protein synthesis with cycloheximide (CHX) decreased this half-life to 30 min without changes in the rate of mrfms gene transcription. In addition oncogenic transformation of L6 alpha 1 myoblasts by the v-fms induced constitutive upregulation of mrfms mRNAs, and nuclear run-on assays demonstrated that mrfms transcription was not growth-factor dependent. Furthermore, these findings with others previously published indicate that mrfms gene products may play a role in the normal and neoplastic growth of muscular cells.

A rat adult skeletal muscle probe (Asm15) originated from a rhabdomyosarcoma was used to isolate the human homologous sequence from a placenta cDNA library. Among several positive clones the longest EcoRI-EcoRI insert (ASM1) obtained was 1875 bp long with 72% homology with rat Asm15 cDNA sequence. Important variations of ASM1 RNA level were observed in different adult skeletal muscles. Expression of a 29kD ASM1 protein was demonstrated in human adult skeletal muscle lysates using an antiserum (PB1579) raised against the C terminal region of the rat Asm15 protein. The human ASM gene was assigned by somatic cell analysis with human (ASM1) and rat (Asm15) probes to chromosome 11, and by in situ hybridization with the human probe to 11p15, a chromosome region involved in human embryonal rhabdomyosarcomas. Except for the presence of a HindII restriction site, the results obtained for the restriction map and the sequence of ASM1 cDNA (data not shown) exhibited extensive homology with the human H19 DNA sequence which have been mapped with a mouse probe also in 11p15. This suggests that ASM/Asm and H19 may represent the same sequence (in this hypothesis the presence of the supplementary HindII site in our ASM1 probe is explained by polymorphic variability). However it was reported that human and mouse H19 mRNA did not encode for a protein but acted as an RNA molecule whereas in our present study ASM protein was detected in human adult skeletal muscle. This could be explained by important regulation of ASM protein expression during development and cell differentiation. However we cannot exclude for the different species studied (mouse, rat, and man) the hypothesis that H19 and ASM/Asm mRNA may represent two distinct messengers from the same gene or even from duplicated genes.

Previously we reported that c-mos proto-oncogene RNA was developmentally up-regulated during post-natal maturation of the rat skeletal muscle. Using two different site-directed affinity-purified antipeptide antibodies we can observe that c-mos product (p43 c-mos) accumulates increasingly during post-natal development of the skeletal muscle and exhibits protein kinase activity. We find that in adult rat p43 c-mos is 10-fold higher in skeletal muscle than in ovaries, and 20- to 40-fold higher than in heart, lung, testis and liver, and may represent about 0.005% of the total soluble proteins. In addition adult skeletal muscle from Xenopus, mouse and man was found to contain p43 c-mos. These data argue in favour of a novel muscle-specific function of c-mos.

We have isolated and sequenced a cDNA clone, homologous to the rat c-mos gene, from a cDNA library of rat skeletal muscles. The 3220 nucleotide cDNA clone codes for a protein of 339 amino acids (37.4 kDa). Both the nucleotide sequence and the deduced amino acid sequence show 60-90% overall homology to Xenopus, chicken, mouse and human mos. By Northern blot analysis, we detected two c-mos transcripts, one major of about 3.6 Kb long, and one minor of about 1.7 Kb long. These are differently regulated during the development of cardiac and skeletal muscles. By Western blot with two antibodies directed against two different portions of the mos protein, we observed in rat muscle two polypeptides of 43 kDa, and 75 kDa respectively.

We previously derived, from a nonmalignant clonal line of rat myogenic cells (L6 alpha 1), two sublines which have lost the capacity to differentiate, the M4 cell of low malignancy and the RMS4 cell of high malignancy. In the present study it is shown that 14 of 15 protooncogenes analyzed exhibit detectable levels of transcripts during L6 alpha 1 cell proliferation. When L6 alpha 1 cells from myotubes, the levels of c-abl, c-myb, and c-Ha-ras transcripts remain unchanged, the level of c-N-ras RNA is augmented, the level of c-erbB RNA is markedly reduced, and all other c-onc transcripts (c-erbA, c-sis, c-src, c-fes, c-fms, c-fos, c-myc, c-Ki-ras, and the putative tyrosine kinase transcript of the c-fgr gene) become hardly, if at all, detectable. Surprisingly, when the three cell types are growing at similar rates only, one protooncogene (c-mos) is not expressed at detectable levels in L6 alpha 1, two others (c-fos, c-erbA) are not expressed in M4 or in RMS4, and three additional ones (c-erbB, c-sis, c-src) are expressed in M4 but not in RMS4. Moreover the level of c-fes RNAs is markedly lower in RMS4 than in M4 or L6 alpha 1. By contrast, the level of two c-Ki-ras 5.4- and 2.2-kilobase transcripts is lower in M4 and L6 alpha 1 than in RMS4, and the latter contains another abundant c-Ki-ras 3.8-kilobase transcript which is hardly detectable in M4 and not at all in L6 alpha 1. These data suggest an activation of the c-Ki-ras gene in the malignant myoblasts and some relationship between the progression of malignancy and inactivation of certain other c-onc genes.

Sodium butyrate, when added in millimolar concentration to a culture of myoblasts of the L6 cell line, inhibits reversibly cell proliferation and differentiation. In the present work, we have studied the effect of Na butyrate on the translational efficiency of the overall poly (A)+ RNA. The mRNA from treated cells was translated in vitro as efficiently as proliferating myoblasts mRNA, while a decrease of translation efficiency was observed with myotubes mRNA. In addition this RNA directs the synthesis of several new polypeptides. on the switch on of alpha actin and myosin heavy chains (MHC), muscle specific genes by the dot blot and Northern blot techniques using cloned probes. Na butyrate prevented the expression of MHC and allowed the switch on of alpha actin gene but at a lesser extent than in normal myotubes. In addition the drug prevented the translocation of alpha actin mRNA into the cytoplasm.

Single-stranded DNA (ssDNA) mainly consisting of transcription sites was probed with cDNA actin clones for studying the expression of actin specific genes during myogenesis in the L6 line of rat myogenic cells. As compared with total nuclear DNA, ssDNA from myoblasts and myotubes was found greatly enriched in sequences complementary to both muscular and non muscular actin sequences. In contrast, ssDNA from spleen, hepatocytes or hepatoma cells was found enriched only in sequences complementary to non muscle actin cDNA. Actin specific sequences accumulated in the ssDNA fraction are almost entirely derived from the coding DNA strand. The DNAase I sensitivity of the actin genes sequences in myogenic and non myogenic cells correlated the data obtained with the ssDNA fraction. It is concluded that the muscle specific actin genes is either transcriptionally active or at least exist in an active configuration in the proliferating myoblasts as well as in the terminally differentiated myotubes.

A nonfusing variant M4 of the L6 line of rat myoblasts was isolated. This subline was tumorigenic when inoculated into suckling rats. Comparative studies of cells growth in vitro, serum requirement, cell adhesiveness and capacity to form colonies in soft agar confirmed that M4 cells had undergone neoplastic transformation while L6 cells exhibited normal characteristics. Another subline designated as RMS4 was derivated from M4 cells tumors. Growth characteristics in vitro and karyotype analysis indicated that RMS4 cells presented a higher degree of transformation as compared with M4 cells. Both M4 and RMS4 cell cultures seem to produce a C-type virus.

Triiodothyronine (T3), injected daily into rats, induces heart hypertrophy. In a recent work we have shown that a single T3 injection stimulates RNA synthesis and modifies the translational products of myocardial mRNAs in reticulocyte lysates, quantitatively and qualitatively. In this work we show that T3 induces small but significant changes in mRNA size distribution after 4 h and much more important changes after 18 h. It also modifies the size distribution of their poly(A) tails. We studied the early effects of this compound on mRNA complexity, using the nucleic acid hybridization technique with DNA complementary to poly(A)+ RNAs. T3, 4 h after injection, suppresses approximately 15% of the sequences, mostly among rare sequences, and increases the frequency of the abundant sequences and of the sequences of intermediary abundance. A large part of this effect disappears 18 h after the injection. It may be concluded that T3 presents an early effect on gene expression, involving changes either at the gene level or and at some post transcriptional level.

Two malignant sublines, M4 and RMS4 , were previously derived from the recloned L6 line of rat myogenic cells. Comparative studies in tissue culture and inoculation into suckling rats indicated that M4 cells and RMS4 cells may be considered as low-malignant and high-malignant cells, respectively, while L6 cells are not malignant. In the present work, we used extracts from L6 cells, M4 cells, and RMS4 cells collected during the period of exponential growth, to compare their polyadenylic acid-containing messenger RNA (mRNA) populations and the corresponding cell-free translation products. Analysis of the hybridization kinetics between radioactive complementary DNA and homologous or heterologous cellular RNAs indicated that L6 cells contained about 28,000 distinct polyadenylic acid-containing mRNA sequences of 1.8 kilobases each, of which 2,000 to 2,500 and 4,000 to 5,000 were missing (or at least were very infrequent) in M4 cells and RMS4 cells, respectively. Using a minor fraction of the RMS4 cell complementary DNAs, partially purified through repeated complementary DNA-RNA hybridization cycles, it was further shown that RMS4 cells contained at least 700 to 800 distinct mRNA species, mainly belonging to the class of low abundance, which appeared to be absent in L6 cells. Most of these mRNA species were also found with a lower frequency in M4 cells. Bidimensional analysis of the cell-free translation products directed by polyadenylic acid-containing mRNA revealed some remarkable differences, in particular the synthesis in a RMS4 cell extract of at least three major polypeptides, possibly related either to the neoplastic process itself or to the stage of malignant transformation.

Sodium butyrate, when added in millimolar concentrations to a culture of myoblasts of the L6 cell line, inhibits reversibly cell proliferation and differentiation. The aim of this work was to study the effect of sodium butyrate on the nuclear and cytoplasmic RNAs in these cells. We have prepared (3H) DNAs complementary to cytoplasmic polyadenylated RNAs from treated and untreated cells and performed homologous and heterologous hybridizations with cytoplasmic polyadenylated RNAs and with total nuclear RNAs. The hybridization kinetics led to the following conclusions: a) Hybridization with nuclear RNAs shows that butyrate allows the transcription of most of the RNA sequences synthesized in proliferating myoblasts, including the sequences that are no longer synthesized in untreated myotubes. However some differences in the hybridization saturation levels indicate that sodium butyrate might modify the expression of a limited number of genes involved in cell proliferation, muscular differentiation, or both. b) Hybridization with cytoplasmic polyadenylated RNAs shows that sodium butyrate acts also at a post-transcriptional level, it produces a large decrease in the frequency of the abundant sequences present in untreated cells, but has little effect on the total number of different RNA species.

Complementary DNAs (cDNAs) were synthesized from polyadenylated RNAs of myoblasts and myotubes and used to analyze changes in the sequence complexity and frequency distribution of messenger RNAs during myogenesis in vitro. cDNA . polyadenylated-RNA hybridization kinetics show the presence of messenger RNA sequences specific for myotubes in fully differentiated muscle cultures. These sequences are accumulated just prior to fusion, as was shown by hybridizations of myotube cDNA and total cytoplasmic RNAs from cells at different stages of differentiation. The myotube cDNA can be enriched 10-fold in myotube-specific RNA species by a hybridization with cytoplasmic RNAs from myoblasts and subsequent removal of these hybridized sequences by hydroxyapatite.

Time course analyses of various proto-oncogene transcripts compared with cytoskeleton-specific and muscle-specific messenger RNAs (mRNAs) were carried out during growth and differentiation of a clonal line of rat myoblasts that retain the capacity to form non-contractile fibres in vitro. Throughout their growth phase, these cells express consistent levels of c-fos, c-myc, c-Ki-ras and c-N-ras RNA and no c-mos RNA. When the cultures approach confluency the level of c-fos RNA rises sharply 3-4-fold, peaks, and rapidly declines when muscle-specific transcripts start accumulating, to become negligible in myotube-forming cells. These changes occur whatever the concentration in seric factors. By contrast, the level of c-N-ras RNA rises up to 3-fold and both c-myc and c-Ki-ras RNAs are slowly eliminated during the myogenic process, whereas no c-mos RNA is detectable. However, skeletal muscles from prenatal fetuses and adult animals were reproducibly found to contain both low and high levels of c-mos RNA respectively. These data and the demonstration that inactivation of the c-fos gene correlates with the loss of myogenic capability in six lines of neoplastic myoblasts, including four lines transformed by the v-fos oncogene, suggest a physiological function for this proto-oncogene during early stages of myogenesis and for the c-N-ras and c-mos genes in later stages of muscular development.

Gene expression is in great part under the control of proteins associated with DNA: histones which can be phosphorylated and acetylated, non-histone proteins which include enzymes, hormone receptors and other regulatory proteins. Phosphorylation seems to play an important role in chromatin regulation.

We previously reported that consistent levels of c-fms-related transcripts are expressed during the growth of rat myogenic cells as well as in all neoplastic myoblasts. The present study extends this observation to mouse myogenic cells and demonstrates that a tyrosine-kinase-associated gp170, very similar or identical to the receptor for the macrophage stimulating factor CSF-1, is synthesized in myoblasts via a short-lived precursor of 115-116 kD and an immature gp130. These gene products are eliminated during the myogenic process, suggesting their role in the proliferation of muscular cells.

Single-stranded DNA (ssDNA), equivalent to about 2% of the total nuclear DNA, was isolated by an improved method of hydroxyapatite chromatography from native nuclear DNA of rat myoblast cells and myotubes of the L6 line. Small quantities of 125I-labelled ssDNA were annealed with a large excess of unlabelled DNA, cytoplasmic RNA and mRNA from myoblasts or myotubes. The results indicated that ssDNA belongs to the non-repetitious portion of the cell genome and is formed of two distinct molecular fractions. The major ssDNA fractions (75%) consist of non-self-reassociating DNA sequences and the minor fraction (25%) consists of self-reassociating DNA sequences. About 30--32% and 25--26% of ssDNA from myoblast represent DNA sequences complementary to total cytplasmic RNAs and polyadenylated RNAs respectively. Hybridizations of ssDNA with an excess of RNA from myoblasts and/or myotubes show differences in the abundance and the diversity of mRNA during mascular differentiation. These differences were confirmed by DNA-driven reactions between 125I-labelled polyadenylated RNA and ssDNA in great excess.

Analyses of intracellular RNAs and proteins, measurements of reverse transcriptase activity and electron microscopy showed that non transformed L6 alpha 1 rat myogenic cells produce no endogenous C-type virus whereas two non fusing variants, the low malignant M4 cell and high malignant RMS4 cell produce small and large amounts of virus respectively consistent with different levels of intracellular viral transcripts. Two non viral transcripts bearing LTR sequences are present in M4 and RMS4 cells and absent from L6 alpha 1 cells although the 3 cell lines exhibit strictly similar patterns of C-type proviral genes.