p57(Kip2) stabilizes the MyoD protein by inhibiting cyclin E-Cdk2 kinase activity in growing myoblasts.

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.

Mos activates myogenic differentiation by promoting heterodimerization of MyoD and E12 proteins.

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.

Differential expression of protooncogenes related to transformation and cancer progression in rat myoblasts.

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.

Accumulation of the c-mos protein is correlated with post-natal development of skeletal muscle.

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.

p34cdc2 protein is complexed with the c-mos protein in rat skeletal muscle.

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.

Dimerization of the amino terminal domain of p57Kip2 inhibits cyclin D1-cdk4 kinase activity.

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.

Direct relationship between the expression of tumor suppressor H19 mRNA and c-mos proto-oncogene during myogenesis.

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.

Isolation and characterization of a cDNA clone encoding for rat CSF-1 gene. Post-transcriptional repression occurs in myogenic differentiation.

A major CSF-1 (Colony-Stimulating Factor 1) mRNA 4.0 kb long was expressed during the proliferation of the L6 alpha 1 rat myogenic cells and was down-regulated after their differentiation into myotubes. A complete cDNA encoding the rat CSF-1 gene (rmCSF-1) was isolated from a cDNA library of L6 alpha 1 myoblasts and sequenced. The overall deduced amino acid sequence was 100% and 68% identical to the mouse and human CSF-1, respectively. While the previously reported mechanisms about the regulation of CSF-1 expression in TPA-treated-monocytes (Horiguchi, J., Sariban, E. and Kufe, D. (1988) Mol. Cell. Biol. 8, 3951-3954) and in fibroblasts (Falkenburg, J.H.F., Harrington, M.A., De Paus, R.A., Walsh, M.K., Daub, R., Landegent, J.E. and Broxmeyer, H.E. (1991) Blood 78, 658-665) involved a control at the transcriptional level, in contrast, the CSF-1 mRNA (half-life approximately 3 h in L6 alpha 1 myoblasts) was post-transcriptionally down-regulated during myogenesis. Inhibition of protein synthesis with cycloheximide (CHX) increased differentially the half-life of CSF-1 mRNA in L6 alpha 1 myotubes compared to L6 alpha 1 myoblasts. Finally, L6 alpha 1 myoblasts were shown to synthesize a 140 kDa homodimeric form of CSF-1. Thus, these findings, together with other results, indicate that CSF-1 gene products may play a role in the normal and neoplastic proliferation of muscular cells.

Overexpression of Mos(rat) proto-oncogene product enhances the positive autoregulatory loop of MyoD.

The myogenic b-HLH transcription factor MyoD activates expression of muscle-specific genes and autoregulates positively its own expression. Various factors such as growth factors and oncogene products repress transcriptional activity of MyoD. The c-mos proto-oncogene product, Mos, is a serine/threonine kinase that can activate myogenic differentiation by specific phosphorylation of MyoD which favors heterodimerization of MyoD and E12 proteins. Here we show that overexpression of Mos enhances the expression level of MyoD protein in myoblasts although phosphorylation of MyoD by Mos does not modify its stability but promotes transcriptional transactivation of the MyoD promoter linked to the luciferase reporter gene. Moreover, co-expression of MyoD with Mos(wt) but not with the kinase-inactive Mos(KM) greatly enhances expression of endogenous MyoD protein and the DNA binding activity of MyoD/E12 heterodimers in 10T1/2 cells. Our data suggest that Mos increases the ability of MyoD to transactivate both muscle-specific genes and its own promoter and could therefore participate in the positive autoregulation loop of MyoD and muscle differentiation.

Ex vivo expansion of megakaryocyte precursor cells in autologous stem cell transplantation for relapsed malignant lymphoma.

To evaluate the impact of ex vivo expanded megakaryocyte (MK) progenitors on high-dose chemotherapy-induced thrombocytopenia, we conducted a phase II study in 10 patients with relapsed lymphoma. Two fractions of peripheral blood progenitor cells (PBPC) were cryopreserved, one with enough cells for at least 2 x 10(6) CD34+ cells/kg and a second obtained after CD34+ selection. Ten days before autologous stem cell transplantation, the CD34+ fraction was cultured with MGDF+SCF for 10 days. After BEAM (BCNU, cyclophosphamide, cytarabine, and melphalan) chemotherapy, patients were reinfused with standard PBPC and ex vivo expanded cells. No toxicity was observed after reinfusion. The mean fold expansion was 9.27 for nucleated cells, 2 for CD34+ cells, 676 for CD41+ cells, and 627 for CD61+ cells. The median date of platelet transfusion independence was day 8 (range: 7-12). All patients received at least one platelet transfusion. In conclusion, ex vivo expansion of MK progenitors was feasible and safe, but this procedure did not prevent BEAM-induced thrombocytopenia. Future studies will determine if expansion of higher numbers of CD34+ cells towards the MK-differentiation pathway will translate into a functional effect in terms of shortening of BEAM-induced thrombocytopenia.

Levobupivacaine hydrochloride and sufentanil have no antimicrobial effect at 25 degrees C in vitro.

BACKGROUND AND OBJECTIVES: Levobupivacaine in combination with sufentanil may be used for labour or postoperative regional analgesia. The risk of bacterial growth within these contained solutions for several hours at room temperature is unknown. We investigated the in vitro antimicrobial effect of levobupivacaine and sufentanil against common micro-organisms encountered during regional anaesthesia. METHODS: Standardized suspensions of Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli were incubated for 1, 3, 6 and 24 h at 25 degrees C, with saline (as control), sufentanil 0.5 or 0.75 microg mL-1, levobupivacaine hydrochloride 5.6 mg mL-1 and concentrations of 1.4, 2.8 and 5 mg mL-1 of levobupivacaine hydrochloride with sufentanil 0.5 microg mL-1. Colony counts were compared after 24 h incubation at 37 degrees C. RESULTS: No bacterial growth was observed on any bacterial strain for any solution tested throughout the experiment. CONCLUSIONS: Our results suggest that solutions of levobupivacaine combined with sufentanil may be used for 24 h at room temperature during regional anaesthesia with no risk of bacterial growth.

Small RNA regulators and the bacterial response to stress.

Recent studies have uncovered dozens of regulatory small RNAs in bacteria. A large number of these small RNAs act by pairing to their target mRNAs. The outcome of pairing can be either stimulation or inhibition of translation. Pairing in vivo frequently depends on the RNA-binding protein Hfq. Synthesis of these small RNAs is tightly regulated at the level of transcription; many of the well-studied stress response regulons have now been found to include a regulatory RNA. Expression of the small RNA can help the cell cope with environmental stress by redirecting cellular metabolism, exemplified by RyhB, a small RNA expressed upon iron starvation. Although small RNAs found in Escherichia coli can usually be identified by sequence comparison to closely related enterobacteria, other approaches are necessary to find the equivalent RNAs in other bacterial species. Nonetheless, it is becoming increasingly clear that many if not all bacteria encode significant numbers of these important regulators. Tracing their evolution through bacterial genomes remains a challenge.

Osteopontin is upregulated by BCR-ABL.

Chronic myelogenous leukemia (CML) is characterized by its hallmark oncogene BCR-ABL and the progression from a chronic phase toward an acute leukemia, with a differentiation arrest of the leukemic clone. In the present study, we conducted a microarray analysis using an inducible model of BCR-ABL expression based on the TET-OFF system, and we found that osteopontin (OPN), a component of stem cell niche, is overexpressed in BCR-ABL-expressing cells. Studies using mutant forms of BCR-ABL demonstrated that the BCR-ABL-induced OPN overexpression was a tyrosine kinase-dependent event. Furthermore, OPN concentration was significantly increased in the serum of leukemic mice generated by transplantation of BCR-ABL-expressing bone marrow cells. Most importantly, a significant increase of OPN concentration was observed in the serum of CML patients as compared to controls. Overall these results show that OPN is deregulated by BCR-ABL oncogene and suggest that OPN could be involved in CML stem cell biology.

Abundant messenger RNA sequences common to transplanted mouse tumours of different tissue origins.

Radioactive cDNA, cell-free-synthesised on poly(A)-rich mRNAs from mouse FLS-ascites tumour cells, were separated into two fractions corresponding to abundant mRNA species and to rare mRNA species respectively, the former being nearly 100-times more abundant per cell than the latter. Aliquots of each fraction were hybridized to a large excess of RNA of different origins; three ascites tumours (the FLS, Krebs 2 and Ehrlich tumours) all transplanted in animals; one established line of Friend erythroleukemia cells serially passaged in culture; adult organs and whole mouse embryos. It was shown that all or nearly all the mRNA species, in particular the abundant ones, were present and similarly distributed in the three transplanted tumour strains studied, despite their different tissue origins. The great majority of abundant mRNA species were also present in Friend erythroleukemia cells but about two-times less frequent relative to transplanted tumours. Much greater differences were found as compared with non-malignant cells. In particular, although the majority of the abundant mRNA species were also found in all normal tissues studied, they were on the average 7-8-times less abundant in adult liver or in 10-days-old embryos, and 10-times less abundant in adult brain, than in tumour cells. Finally about 10% of the abundant RNA species common to all transplanted tumours appeared to be absent in normal cells, whatever their growth rates or stage of differentiation. The possible implications of these findings are discussed.

Identification of a cis acting element responsible for muscle specific expression of the c-mos protooncogene.

A series of deletion constructs of the 5' flanking region of rat c-mos gene was positioned upstream to the CAT gene and transfected into muscle and non-muscle cells. CAT activities revealed that a region located downstream of a TATA box and containing the proximal transcription start site is the muscle c-mos promoter. This promoter is more efficient in L6 alpha 1 myoblasts than in L6 alpha 1 myotubes but not in C3H10T1/2 cells. Gel shift assays demonstrated that nuclear proteins from myoblasts and myotubes formed complexes migrating differently. Footprinting analyses showed that nuclear proteins from L6 alpha 1 myoblasts protected a DNA fragment located at position nt -979 to nt -938 relative to the first ATG of the rat c-mos ORF while nuclear proteins from myotubes protected the DNA between nt -998 to nt -928. Furthermore one of protein - DNA complexes containing the proximal transcription start site, included a consensus sequence TGTC(AGT/TCG)CC(A/T)G present in the initiator element (Inr) of several genes. Southwestern blot analysis pointed to a 82kDa polypeptide as a potential candidate for trans acting factor in myoblasts. In L6 alpha 1 myotubes this polypeptide is replaced by other proteins of 40-42kDa and 82kDa. An interplay between these two complexes may constitute a developmental as well as a physiologically regulated mechanism that modulates c-mos expression during the early stages of myogenesis.

Possible role of c-fos, c-N-ras and c-mos proto-oncogenes in muscular development.

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.

Expression of extracellular matrix genes in relation to myogenesis and neoplastic transformation.

Fibronectin and alpha 1(I) and alpha 2(I) collagen proteins and RNAs are highly expressed during the growth phase of the non-transformed L6 alpha 1 rat myoblasts. When L6 alpha 1 cells from myotubes following transfer to low serum medium, the levels of fibronectin RNA decrease 8-fold, those of both alpha 2(I) transcripts decrease only 2-fold, while those of both alpha 1(I) transcripts remain stable. The L6 alpha 1 cell-derived non-differentiable low-malignant M4 cell and high-malignant RMS4 cell display only one size of alpha 1(I) and alpha 2(I) transcripts. Compared with L6 alpha 1 myoblasts, the levels of fibronectin and alpha 1(I) RNAs are reduced by factors of 4-5 and 9-10 respectively in both M4 and RMS4 and those of alpha 2(I) RNAs by factors of 10-11 and 20-22 in M4 and RMS4, respectively. Transcription rates are similarly decreased for fibronectin RNA, but are decreased less for collagen RNAs.

Molecular cloning of CSF-1 receptor from rat myoblasts. Sequence analysis and regulation during myogenesis.

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.

Evidence of ABL-kinase domain mutations in highly purified primitive stem cell populations of patients with chronic myelogenous leukemia.

To study the hierarchical levels of stem cell targets for ABL-kinase domain mutations in CML, highly purified CD34+CD38- and CD34+CD38+ cell populations and their LTC-IC-derived progeny were analyzed in four patients at diagnosis (n=1) or in advanced phases (n=3) of their disease. In the single patient with early phase CML who later developed an Imatinib Mesylate-resistance and a Y253H mutation, no mutation was detectable in purified cell fractions analyzed at diagnosis nor in their LTC-IC-derived progeny. In contrast, in three patients in advanced phase CML, ABL-kinase mutations demonstrated in peripheral blood cells by sequencing (Q252E and M351T) were detectable in the FACS-sorted cells and became amplified in the LTC-IC-derived progeny of the primitive cells. These findings demonstrate that in late CP or advanced CML, ABL-kinase mutations occur as an intraclonal event in the primitive Ph1+ stem cell compartments with progression of this clone towards IM-resistant blast phase.