Marie-Pierre Leibovitch

The human ASM (adult skeletal muscle) gene: expression and chromosomal assignment to 11p15

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.

Effect of sodium butyrate on myoblast growth and differentiation

Abstract Myoblasts from L6 line, after a period of cell division, undergo differentiation into large multinucleated syncitia in 8–9 days. Butyrate was added for 24 hours at various times of culture. In all samples growth was strongly inhibited. After removal of butyrate, growth continued at the same rate for 2 days, afterwards the growth rate became the same as in control cells. Morphological and biochemical differentiations, estimated by creatine phosphokinase assay, occur with a 1–3 day delay according to the time of addition of butyrate, when compared to untreated cells. Only the M form of creatine phosphokinase was present in butyrate-treated cells as in untreated myoblasts.

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 α-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.

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.

Mutation of MyoD-Ser237 abolishes its up-regulation by c-Mos

Recently we have shown that Mos could activate myogenic differentiation by promoting heterodimerisation of MyoD and E12 proteins. Here, we demonstrate that MyoD can be efficiently phosphorylated by in vitro kinase assay with purified Mos immunoprecipitated from transfected cells. Comparative two‐dimensional tryptic phosphopeptide mapping combined with site‐directed mutagenesis revealed that Mos phosphorylates MyoD on serine 237. Mutation of serine 237 to a non‐phosphorylable alanine (MyoD‐Ala237) abolished the positive regulation of MyoD by Mos following overexpression in proliferating 10T1/2 cells. Taken together, our data show that direct phosphorylation of MyoD‐Ser237 by Mos plays a positive role in increasing MyoD activity during myoblast proliferation.

Proto‐oncogenes and Differentiation versus Transformation of Striated Muscle Cells

It would be a truism to recall that unrestricted cell proliferation is a necessary but not sufficient condition for malignant transformation. Besides the crucial role of host factors, a multitude of observations demonstrate that cancer cells display variable neoplastic properties and are generally more-or-less dedifferentiated, whereas they often express anachronistic or ectopic gene products (see Refs. 1 and 2 for review). The concept that misprogramming of genetic information is operational in carcinogenesis is indeed an old one, but it remained essentially speculative until the late sixties. In 1971 a successful approach to verify this concept was opened by the pioneer work of Charlotte Friend and her colleagues, who showed that erythroid differentiation of Friend erythroleukemia cells was induced by addition of dimethyl sulfoxide.* Further advances were rendered possible by the rapid development of research on proto-oncogenes (or c-onc genes) initially characterized as the cellular counterparts of the transforming (viral) genes (v-onc) of oncogenic retroviruses (see Ref. 6 for review). Over the last few years, a growing body of evidence suggests that many of the proto-oncogenes identified thus far (possibly all of them) are normally involved in cell proliferation, development, and differentiation. For example, the proteins encoded by the c-sis, c-fms, c-erB and c-erbA genes are related, respectively, to platelet-derived growth factor (PDGF),’.’ the cell surface receptor for the macrophage growth factor CSF-1,9 the cell surface receptor for epithelial growth factor (EGF),’’ and the nuclear receptor for thyroid hormone.”,” Expression of the ~-myc‘~-’’ and ~ -Ki r a s ’~ . ’~ genes is enhanced when quiescent cells in culture are induced to proliferate or when liver is regenerating.” A rapid but transient expression of the c-fos proto-oncogene precedes