Absence Of New Protein Synthesis Research Articles

Wnt, GSK3, and Macropinocytosis.

Here we review the regulation of macropinocytosis by Wnt growth factor signaling. Canonical Wnt signaling is normally thought of as a regulator of nuclear β-catenin, but emerging results indicate that there is much more than β-catenin to the Wnt pathway. Macropinocytosis is transiently regulated by EGF-RTK-Ras-PI3K signaling. Recent studies show that Wnt signaling provides for sustained acquisition of nutrients by macropinocytosis. Endocytosis of Wnt-Lrp6-Fz receptor complexes triggers the sequestration of GSK3 and components of the cytosolic destruction complex such as Axin1 inside multivesicular bodies (MVBs) through the action of the ESCRT machinery. Wnt macropinocytosis can be induced both by the transcriptional loop of stabilized β-catenin, and by the inhibition of GSK3 even in the absence of new protein synthesis. The cell is poised for macropinocytosis, and all it requires for triggering of Pak1 and the actin machinery is the inhibition of GSK3. Striking lysosomal acidification, which requires macropinocytosis, is induced by GSK3 chemical inhibitors or Wnt protein. Wnt-induced macropinocytosis requires the ESCRT machinery that forms MVBs. In cancer cells, mutations in the tumor suppressors APC and Axin1 result in extensive macropinocytosis, which can be reversed by restoring wild-type protein. In basal cellular conditions, GSK3 functions to constitutively repress macropinocytosis.

Synchronized HIV assembly by tunable PIP2 changes reveals PIP2 requirement for stable Gag anchoring.

HIV-1 assembles at the plasma membrane (PM) of infected cells. PM association of the main structural protein Gag depends on its myristoylated MA domain and PM PI(4,5)P2. Using a novel chemical biology tool that allows rapidly tunable manipulation of PI(4,5)P2 levels in living cells, we show that depletion of PI(4,5)P2 completely prevents Gag PM targeting and assembly site formation. Unexpectedly, PI(4,5)P2 depletion also caused loss of pre-assembled Gag lattices from the PM. Subsequent restoration of PM PI(4,5)P2 reinduced assembly site formation even in the absence of new protein synthesis, indicating that the dissociated Gag molecules remained assembly competent. These results reveal an important role of PI(4,5)P2 for HIV-1 morphogenesis beyond Gag recruitment to the PM and suggest a dynamic equilibrium of Gag-lipid interactions. Furthermore, they establish an experimental system that permits synchronized induction of HIV-1 assembly leading to induced production of infectious virions by targeted modulation of Gag PM targeting.

Matrix as an Interstitial Transport System

The extracellular matrix (ECM) is best known for its function as a structural scaffold for the tissue and more recently as a microenvironment to sequester growth factors and cytokines allowing for rapid and localized changes in their activity in the absence of new protein synthesis. In this review, we explore this and additional new aspects of ECM function in mediating cell-to-cell communications. Fibrillar and nonfibrillar components of ECM can limit and facilitate the transport of molecules through the extracellular space while also regulating interstitial hydrostatic pressure. In turn, transmembrane communications via molecules, such as ECM metalloproteinase inducer, thrombospondins, and integrins, can further mediate cell response to extracellular cues and affect ECM composition and tissue remodeling. Other means of cell-to-cell communication include extracellular microRNA transport and its contribution to gene expression in target cells and the nanotube formation between distant cells, which has recently emerged as a novel conduit for intercellular organelle sharing thereby influencing cell survival and function. The information summarized and discussed here are not limited to the cardiovascular ECM but encompass ECM in general with specific references to the cardiovascular system.

Transcription, Splicing, and Release: Are We There Yet?

A systematic analysis of LPS-induced gene expression in macrophages by Bhatt et al. demonstrates that inflammatory responses are governed primarily at the level of transcription initiation. Unexpectedly, full-length nascent RNAs that contain introns appear to accumulate on chromatin, presumably to complete processing, prior to release of functional mRNA for export to the cytoplasm.

USP1 Deubiquitinates ID Proteins to Preserve a Mesenchymal Stem Cell Program in Osteosarcoma

Inhibitors of DNA binding (IDs) antagonize basic-helix-loop-helix (bHLH) transcription factors to inhibit differentiation and maintain stem cell fate. ID ubiquitination and proteasomal degradation occur in differentiated tissues, but IDs in many neoplasms appear to escape degradation. We show that the deubiquitinating enzyme USP1 promotes ID protein stability and stem cell-like characteristics in osteosarcoma. USP1 bound, deubiquitinated, and thereby stabilized ID1, ID2, and ID3. A subset of primary human osteosarcomas coordinately overexpressed USP1 and ID proteins. USP1 knockdown in osteosarcoma cells precipitated ID protein destabilization, cell-cycle arrest, and osteogenic differentiation. Conversely, ectopic USP1 expression in mesenchymal stem cells stabilized ID proteins, inhibited osteoblastic differentiation, and enhanced proliferation. Consistent with USP1 functioning in normal mesenchymal stem cells, USP1-deficient mice were osteopenic. Our observations implicate USP1 in preservation of the stem cell state that characterizes osteosarcoma and identify USP1 as a target for differentiation therapy.

102. CHARACTERISATION OF THE GTPase DYNAMIN THROUGHOUT MURINE SPERM MATURATION

Throughout sperm maturation distinct remodelling events occur that imbue the cells with both the ability to bind the zona pellucida and undergo the acrosome reaction. Of long standing interest to our laboratory is the elucidation of the molecular mechanisms that underpin the attainment of these key functional attributes. This process begins with a complex range of morphological changes that accompany spermatogenesis, and is continued through post-testicular phases of maturation in both the male (epididymal maturation) and female (capacitation) reproductive tracts. However, among these changes only those occurring during the initial stages of spermatogenesis are intrinsically driven. The fact that the majority of sperm remodelling is extrinsically stimulated, and occurs in the absence of new protein synthesis, highlights the potential importance of processes such as intracellular protein trafficking. This has directed our focus towards the dynamin family of protein traffickers. The GTPase dynamin exists in three isoforms, namely Dnm1, Dnm2 and Dnm3 and is an integral part of the molecular machinery required for vesicle mediated protein translocation. Recent research from our laboratory has demonstrated the presence of these three isoforms in distinct, cell-specific locations during murine spermatogenesis. Immunofluorescence on mouse testis revealed that both Dnm1 and 2 are present within a region corresponding to the developing acrosome in maturing sperm, whilst Dnm3 appears to reside solely within pre-meiotic germ cells. Interestingly, Dnm1 and Dnm2 are both retained within the peri-acrosomal region of the sperm head in mature spermatozoa. Additionally, upon the induction of capacitation in vitro, staining for both Dnm1 and 2 becomes significantly reduced. Collectively these data support the novel hypothesis that dynamin not only participates in sperm remodelling events during spermatogenesis but may also have a previously unappreciated role in capacitation-associated priming of the sperm surface for interaction with the oocyte.

A Role for Checkpoint Kinase-Dependent Rad26 Phosphorylation in Transcription-Coupled DNA Repair in Saccharomyces cerevisiae

Upon DNA damage, eukaryotic cells activate a conserved signal transduction cascade known as the DNA damage checkpoint (DDC). We investigated the influence of DDC kinases on nucleotide excision repair (NER) in Saccharomyces cerevisiae and found that repair of both strands of an active gene is affected by Mec1 but not by the downstream checkpoint kinases, Rad53 and Chk1. Repair of the nontranscribed strand (by global genome repair) requires new protein synthesis, possibly reflecting the involvement of Mec1 in the activation of repair genes. In contrast, repair of the transcribed strand by transcription-coupled NER (TC-NER) occurs in the absence of new protein synthesis, and DNA damage results in Mec1-dependent but Rad53-, Chk1-, Tel1-, and Dun1-independent phosphorylation of the TC-NER factor Rad26, a member of the Swi/Snf group of ATP-dependent translocases and yeast homologue of Cockayne syndrome B. Mutation of the Rad26 phosphorylation site results in a decrease in the rate of TC-NER, pointing to direct activation of Rad26 by Mec1 kinase. These findings establish a direct role for Mec1 kinase in transcription-coupled repair, at least partly via phosphorylation of Rad26, the main transcription-repair coupling factor.

Identification of Direct Thyroid Hormone Response Genes Reveals the Earliest Gene Regulation Programs during Frog Metamorphosis

Thyroid hormone (T3) is essential for normal development and organ function throughout vertebrates. Its effects are mainly mediated through transcriptional regulation by T3 receptor (TR). The identification and characterization of the immediate early, direct target genes are thus of critical importance in understanding the molecular pathways induced by T3. Unfortunately, this has been hampered by the difficulty to study gene regulation by T3 in uterus-enclosed mammalian embryos. Here we used Xenopus metamorphosis as a model for vertebrate postembryonic development to identify direct T3 response genes in vivo. We took advantage of the ability to easily induce metamorphosis with physiological levels of T3 and to carry out microarray analysis in Xenopus laevis and genome-wide sequence analysis in Xenopus tropicalis. This allowed us to identify 188 up-regulated and 249 down-regulated genes by T3 in the absence of new protein synthesis in whole animals. We further provide evidence to show that these genes contain functional TREs that are bound by TR in tadpoles and that their promoters are regulated by TR in vivo. More importantly, gene ontology analysis showed that the direct up-regulated genes are enriched in categories important for transcriptional regulation and protein degradation-dependent signaling processes but not DNA replication. Our findings thus revealed the existence of interesting pathways induced by T3 at the earliest step of metamorphosis.

Class-specific Regulation of Pro-inflammatory Genes by MyD88 Pathways and IκBζ

Toll-like receptors trigger the induction of primary response genes via MyD88-mediated activation of NF-kappaB and other transcription factors. These factors then act in concert with primary response gene products to induce secondary response genes. Although the MyD88 pathway is important for the expression of both primary and secondary response genes, we show that the recruitment of NF-kappaB, RNA polymerase, and the TATA-binding protein is MyD88-dependent only at secondary response genes. This selective dependence correlates with the fact that MyD88 is required for nucleosome remodeling and histone H3K4 trimethylation at secondary response promoters, whereas rapidly induced primary response promoters are assembled into poised MyD88-independent chromatin structures. At a subset of secondary response promoters, IkappaBzeta was identified as a selective regulator of H3K4 trimethylation and preinitiation complex assembly after nucleosome remodeling. These mechanistic distinctions advance our understanding of the diverse molecular cascades that underlie the differential regulation of pro-inflammatory genes.

Reduced levels of Ago2 expression result in increased siRNA competition in mammalian cells

Administration of small interfering RNAs (siRNAs) leads to degradation of specific mRNAs utilizing the cellular RNA interference (RNAi) machinery. It has been demonstrated that co-administration of siRNAs may lead to attenuation of activity of one of the siRNAs. Utilizing antisense and siRNA-mediated RNA-induced silencing complex (RISC) gene reduction we show that siRNA competition is correlated with differences in the cellular expression levels of Ago2, while levels of other RISC proteins have no effect on competition. We also show that under certain conditions siRNA competition rather than reduction of cellular RISC levels may be responsible for apparent reduction in siRNA activity. Furthermore, exploiting siRNA competition, we show that the RISC pathway loads and results in detectable cleavage of the target RNA in ∼2 h after transfection. The RISC pathway is also capable of being reloaded even in the absence of new protein synthesis. RISC reloading and subsequent induction of detectable cleavage of a new target RNA, requires about 9–12 h following the initial transfection.

Mitotic accumulations of PML protein contribute to the re-establishment of PML nuclear bodies in G1

Although the mechanism of chromosomal segregation is well known, it is unclear how other nuclear compartments such as promyelocytic leukemia (PML) nuclear bodies partition during mitosis and re-form in G1. We demonstrate that PML nuclear bodies partition via mitotic accumulations of PML protein (MAPPs), which are distinct from PML nuclear bodies in their dynamics, biochemistry and structure. During mitosis PML nuclear bodies lose biochemical components such as SUMO-1 and Sp100. We demonstrate that MAPPs are also devoid of Daxx and these biochemical changes occur prior to chromatin condensation and coincide with the loss of nuclear membrane integrity. MAPPs are highly mobile, yet do not readily exchange PML protein as demonstrated by fluorescence recovery after photo-bleaching (FRAP). A subset of MAPPs remains associated with mitotic chromosomes, providing a possible nucleation site for PML nuclear body formation in G1. As the nuclear envelope reforms in late anaphase, these nascent PML nuclear bodies accumulate components sequentially, for example Sp100 and SUMO-1 before Daxx. After cytokinesis, MAPPs remain in the cytoplasm long after the reincorporation of splicing components and their disappearance coincides with new PML nuclear body formation even in the absence of new protein synthesis. The PML protein within MAPPs is not degraded during mitosis but is recycled to contribute to the formation of new PML nuclear bodies in daughter nuclei. The recycling of PML protein from one cell cycle to the next via mitotic accumulations may represent a common mechanism for the partitioning of other nuclear bodies during mitosis.

Mammalian αI-spectrin is a neofunctionalized polypeptide adapted to small highly deformable erythrocytes

Mammalian red blood cells, unlike those of other vertebrates, must withstand the rigors of circulation in the absence of new protein synthesis. Key to this is plasma membrane elasticity deriving from the protein spectrin, which forms a network on the cytoplasmic face. Spectrin is a tetramer (alphabeta)(2), made up of alphabeta dimers linked head to head. We show here that one component of erythrocyte spectrin, alphaI, is encoded by a gene unique to mammals. Phylogenetic analysis suggests that the other alpha-spectrin gene (alphaII) common to all vertebrates was duplicated after the emergence of amphibia, and that the resulting alphaI gene was preserved only in mammals. The activities of alphaI and alphaII spectrins differ in the context of the human red cell membrane. An alphaI-spectrin fragment containing the site of head-to-head interaction with the beta-chain binds more weakly than the corresponding alphaII fragment to this site. The latter competes so strongly with endogenous alphaI as to cause destabilization of membranes at 100-fold lower concentration than the alphaI fragment. The efficacies of alphaI/alphaII chimeras indicate that the partial structural repeat, which binds to the complementary beta-spectrin element, and the adjacent complete repeat together determine the strength of the dimer-dimer interaction on the membrane. Alignment of all available alpha-spectrin N-terminal sequences reveals three blocks of sequence unique to alphaI. Furthermore, human alphaII-spectrin is closer to fruitfly alpha-spectrin than to human alphaI-spectrin, consistent with adaptation of alphaI to new functions. We conclude that alphaI-spectrin represents a neofunctionalized spectrin adapted to the rapid make and break of tetramers.

Identification of Direct Genomic Targets Downstream of the Nuclear Factor-κB Transcription Factor Mediating Tumor Necrosis Factor Signaling

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the nuclear factor-kappaB (NF-kappaB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-kappaB-dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-kappaB inhibitor to systematically identify NF-kappaB-dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-kappaB signaling was analyzed. We identified 50 unique genes that were regulated by TNF (Pr(F)<0.001) and demonstrated a change in signal intensity of+/-3-fold relative to control. Of these, 28 were NF-kappaB-dependent, encoding proteins involved in diverse cellular activities. Quantitative real-time PCR assays of eight characterized NF-kappaB-dependent genes and five genes not previously known to be NF-kappaB-dependent (Gro-beta and-gamma, IkappaBepsilon, interleukin (IL)-7R, and Naf-1) were used to determine whether they were directly or indirectly NF-kappaB regulated. Expression of constitutively active enhanced green fluorescent.NF-kappaB/Rel A fusion protein transactivated all but IL-6 and IL-7R in the absence of TNF stimulation. Moreover, TNF strongly induced all 12 genes in the absence of new protein synthesis. High probability NF-kappaB sites in novel genes were predicted by binding site analysis and confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays show the endogenous IkappaBalpha/epsilon, Gro-beta/gamma, and Naf-1 promoters directly bound NF-kappaB/Rel A in TNF-stimulated cells. Together, these studies systematically identify the direct NF-kappaB-dependent gene network downstream of TNF signaling, extending our knowledge of biological processes regulated by this pathway.

Nmyc upregulation by sonic hedgehog signaling promotes proliferation in developing cerebellar granule neuron precursors.

Hedgehog pathway activation is required for expansion of specific neuronal precursor populations during development and is etiologic in the human cerebellar tumor, medulloblastoma. We report that sonic hedgehog (Shh) signaling upregulates expression of the proto-oncogene Nmyc in cultured cerebellar granule neuron precursors (CGNPs) in the absence of new protein synthesis. The temporal-spatial expression pattern of Nmyc, but not other Myc family members, precisely coincides with regions of hedgehog proliferative activity in the developing cerebellum and is observed in medulloblastomas of Patched (Ptch) heterozygous mice. Overexpression of Nmyc promotes cell-autonomous G(1) cyclin upregulation and CGNP proliferation independent of Shh signaling. Furthermore, Myc antagonism in vitro significantly decreases proliferative effects of Shh in cultured CGNPs. Together, these findings identify Nmyc as a direct target of the Shh pathway that functions to regulate cell cycle progression in cerebellar granule neuron precursors.

Inhibition of IL-6 signaling by a p38-dependent pathway occurs in the absence of new protein synthesis

Negative regulation of cytokine signaling is important for limiting the intensity and duration of cytokine action and for maintaining homeostasis. Several constitutive mechanisms for suppressing cytokine Jak-STAT signaling have been described. Inducible or regulated inhibition of cytokine signaling is equally important, and much attention has been focused on inhibition mediated through the induction of expression of suppressors of cytokine signaling (SOCS proteins). We have previously reported IL-1-induced inhibition of IL-6 signaling in monocytes, and herein we use inhibitors of protein synthesis to demonstrate that inhibition of IL-6 signaling can occur in the absence of new protein synthesis. Surprisingly, some protein synthesis inhibitors themselves inhibited IL-6 signaling rapidly, strengthening the conclusion that IL-6 signaling can be inhibited in the absence of protein synthesis. Inhibition of IL-6 signaling by IL-1 and protein synthesis inhibitors was dependent on the p38 stress kinase, and activation of p38 secondary to inducible expression of MKK6 was sufficient to inhibit IL-6 signaling. Inhibition was specific for IL-6, as induction of STAT activation by IFN-gamma, IFN-alpha, and vanadate was not inhibited. IL-1-induced inhibition of IL-6 signaling was not mediated by the activation of tyrosine phosphatases or by p38-dependent activation of phospholipase A(2) or cyclooxygenases, which could lead to indirect inhibition via production of prostaglandins. These results identify an inducible mechanism of inhibition of IL-6 signaling that is direct and independent of induction of negative regulators such as SOCS proteins. A role for p38 in mediating inhibition suggests that multiple cytokines and stress agents that activate p38 pathways in monocytes, such as IL-1, TNF, Toll-like receptors, and Fc receptors, can modulate Jak-STAT signaling by pleiotropic cytokines such as IL-6.

L-Cysteine-mediated Destabilization of Dinitrosyl Iron Complexes in Proteins

Nitric oxide is a signaling molecule in intercellular communication as well as a powerful weapon used by macrophages to kill tumor cells and pathogenic bacteria. Here, we show that when Escherichia coli cells are exposed to nitric oxide, its ferredoxin [2Fe-2S] cluster is nitrosylated, forming the dinitrosyl iron complex with a characteristic EPR signal at g(av) = 2.04. Such formed ferredoxin dinitrosyl iron complex is efficiently repaired in E. coli cells even in the absence of new protein synthesis. However, the repair activity is completely inactivated once E. coli cells are disrupted, indicating that repairing the ferredoxin dinitrosyl iron complex requires cellular reducing equivalents. In search of such cellular factors, we find that l-cysteine can effectively eliminate the EPR signal of the ferredoxin dinitrosyl iron complex and release the ferrous iron from the complex. In contrast, N-acetyl-l-cysteine and reduced glutathione are much less effective. l-Cysteine seems to have a general function, since it can also remove the otherwise stable dinitrosyl iron complexes from proteins in the cell extracts prepared from the E. coli cells treated with nitric oxide. We propose that l-cysteine is responsible for removing the dinitrosyl iron complexes from the nitric oxide-modified proteins into which a new iron-sulfur cluster will be reassembled.

A Constitutive Cytoprotective Pathway Protects Endothelial Cells from Lipopolysaccharide-induced Apoptosis

Lipopolysaccharide (LPS) has been implicated as the bacterial component responsible for much of the endothelial cell injury/dysfunction associated with Gram-negative bacterial infections. Protein synthesis inhibition is required to sensitize the endothelium to lipopolysaccharide-induced apoptosis, suggesting that a constitutive or inducible cytoprotective protein(s) is required for endothelial survival. We have identified two known endothelial anti-apoptotic proteins, c-FLIP and Mcl-1, the expression of which is decreased markedly in the presence of cycloheximide. Decreased expression of both proteins preceded apoptosis evoked by lipopolysaccharide + cycloheximide. Caspase inhibition protected against apoptosis, but not the decreased expression of c-FLIP and Mcl-1, suggesting that they exert protection upstream of caspase activation. Inhibition of the degradation of these two proteins with the proteasome inhibitor, lactacystin, prevented lipopolysaccharide + cycloheximide-induced apoptosis. Similarly, lactacystin protected against endothelial apoptosis induced by either tumor necrosis factor-alpha or interleukin-1beta in the presence of cycloheximide. That apoptosis could be blocked in the absence of new protein synthesis by inhibition of the proteasome degradative pathway implicates the requisite involvement of a constitutively expressed protein(s) in the endothelial cytoprotective pathway. Finally, reduction of FLIP expression with antisense oligonucleotides sensitized endothelial cells to LPS killing, demonstrating a definitive role for FLIP in the protection of endothelial cells from LPS-induced apoptosis.

Active recycling of yeast Golgi mannosyltransferase complexes through the endoplasmic reticulum.

Mnn9p is a component of two distinct multiprotein complexes in the Saccharomyces cerevisiae cis-Golgi that have both been shown to have alpha-1,6-mannosyltransferase activity in vitro. In one of these complexes, Mnn9p associates with four other membrane proteins, Anp1p, Mnn10p, Mnn11p, and Hoc1p, whereas the other complex consists of Mnn9p and Van1p. Members of the Mnn9p-containing complexes were incorporated into COPII vesicles made in vitro from endoplasmic reticulum (ER) membranes isolated from cycloheximide-treated cells. This behavior is consistent with an active Golgi to ER recycling process. To examine this path in vivo, we monitored retrograde transport of subunits of the complex in cells blocked in anterograde transport from the ER. In this situation, specific relocation of the proteins from the Golgi to the ER was observed in the absence of new protein synthesis. Conversely, when retrograde transport was blocked in vivo, subunits of the mannosyltransferase complex accumulated in the vacuole. Packaging of Mnn9p in COPI-coated vesicles from purified Golgi membranes was also investigated using a coatomer-dependent vesicle budding assay. Gradient fractionation experiments showed that Mnn9p and the retrograde v-SNARE, Sec22p, were incorporated into COPI-coated vesicles. These observations indicate that the Mnn9p-containing mannosyltransferase complexes cycle back and forth between the ER and Golgi.

Signaling themes shared between peptide and steroid hormones at the plasma membrane.

Steroid hormone receptors are primarily thought of as transcriptional regulators that are activated by ligand binding. However, there is also evidence for "nongenomic" actions of steroid hormones. Cheryl Watson summarizes a recent meeting that brought together researchers exploring these rapid effects of steroid hormones that occur in the absence of new protein synthesis. Current evidence for the existence of membrane steroid receptors, which may or may not be related to the well-known nuclear receptors, is discussed.

Critical role played by cyclin D3 in the MyoD-mediated arrest of cell cycle during myoblast differentiation.

During the terminal differentiation of skeletal myoblasts, the activities of myogenic factors regulate not only tissue-specific gene expressions but also the exit from the cell cycle. The induction of cell cycle inhibitors such as p21 and pRb has been shown to play a prominent role in the growth arrest of differentiating myoblasts. Here we report that, at the onset of differentiation, activation by MyoD of the Rb, p21, and cyclin D3 genes occurs in the absence of new protein synthesis and with the requirement of the p300 transcriptional coactivator. In differentiated myocytes, cyclin D3 also becomes stabilized and is found nearly totally complexed with unphosphorylated pRb. The detection of complexes containing cyclin D3, cdk4, p21, and PCNA suggests that cdk4, along with PCNA, may get sequestered into high-order structures held together by pRb and cyclin D3. Cyclin D3 up-regulation and stabilization is inhibited by adenovirus E1A, and this correlates with the ability of E1A to promote pRb phosphorylation; conversely, the overexpression of cyclin D3 in differentiated myotubes counteracts the E1A-mediated reactivation of DNA synthesis. These results indicate that cyclin D3 critically contributes to the irreversible exit of differentiating myoblasts from the cell cycle.