François Dautry

c-myc Oncogene expression inhibits the initiation of myogenic differentiation

The role of c-myc oncogene expression in myogenic differentiation has been established by transfecting rat myoblasts of the L6 cell line with plasmid pMT-myc, in which the c-myc coding sequences were under the control of the metallothionein I promoter. We observed that the constitutive expression of the exogenous c-myc gene inhibits muscular differentiation. A diminution of the endogenous c-myc gene expression occurs within the first 24 h after the transfer of the cells to a differentiating medium. This early decrease of c-myc expression is required for cell differentiation to occur. We have also observed that exogenous myc gene expression has no effect on endogenous myc expression.

Regulation of pre-mRNA processing by src

BACKGROUND Changes in gene expression in response to external signals provide a key mechanisms for the regulation of higher eukaryotic cell functions. The importance of transcriptional control in the response of cells to growth factors and cytokines has been extensively documented, but gene expression has also been shown to be controlled at other levels, such as the stability of mRNA in the cytoplasm, its localization and translation. By contrast to transcriptional control, little is known of the contribution of pre-mRNA nuclear processing to the regulation of gene expression, as most of our knowledge of pre-mRNA processing in vivo is indirect, being inferred from comparisons of transcription rates and levels of mRNA accumulation. RESULTS In this study, we have used as a model the well-characterized maturation pathway of transcripts of the cytokine, tumour necrosis factor beta (TNF beta). We have used the murine TNF beta gene as a reporter for pre-mRNA processing, using a co-transfection approach to investigate whether overproduction of proteins involved in signal transduction influences the processing of TNF beta transcripts. Although transfection of both activated ras and src genes led to an increase in RNA accumulation in the nuclear and cytoplasmic compartments, as expected from their transactivation of the TNF beta expression vector, only src induced a modification of RNA processing. Comparison of several modes of src activation indicated that two distinct effects of src on pre-mRNA processing can be coupled: one involves slowing down splicing and the other allows the export of partially spliced transcripts. These effects can be observed not only on the three introns of TNF beta but also on transcripts from a beta globin expression vector. DISCUSSION We have characterized how the processing of transcripts of TNF beta and beta globin is regulated by the signal transduction pathway that includes the Src protein, establishing that external signals have the capacity to regulate gene expression at a post-transcriptional level within the nucleus. Src seems to act on a general mechanism of splicing and/or mRNA transport, but its biologically relevant targets are likely to be restricted to genes for which either alternative processing pathways are in competition, or the kinetics of splicing is critical. This regulation could reflect a modulation by Src of the activity of components of the splicing and transport machineries, but could also involve RNA-binding proteins, which have been shown to interact with Src.

Exon skipping in the expression of the gene immediately upstream of N-ras (unr/NRU)

The unr (or NRU) gene was identified during investigations of the structure of the N-ras locus in the genome of mammals. A striking feature of the unr/N-ras gene tandem is the short intergenic distance of 150 nucleotides which suggests the possibility of transcriptional interactions between the two genes. At present, the function of the unr gene is unknown, but the predicted translation product shows a distant relationship to a class of DNA binding proteins. Comparison of the two published cDNA sequences, from a human lymphocytic and a rat testis cDNA library, reveals a difference of 31 amino acids in the size of the predicted proteins. We show that this is due to the skipping of exon 5 within the human NRU gene and that a similar phenomenon occurs in the rat unr gene. Exon skipping takes place in all the cells and tissues we have analyzed and generates the predominant form of message, except in the brain where both classes are about as abundant. This exon skipping is independent of other aspects of unr expression such as the choice of the polyadenylation site.

Growth stimulation of rat primary embryo fibroblasts by the human myc gene

We have studied the ability of plasmids encoding a normal human myc protein to stimulate growth of primary rat embryo fibroblasts. We measured growth stimulation by the number of G418-resistant colonies obtained after co-transfection with plasmid pSV2neo and by the percentage of these colonies that grew in long-term culture (immortalization). Using a normal human myc gene, we detected a weak growth stimulation at the colony formation stage and a low frequency of immortalization. Replacement of the myc promoter by a heterologous promoter (mouse metallothionein I promoter) and deletion of the first non-coding exon led to a more efficient growth stimulation by both criteria. Thus, disregulation of c-myc is essential for an altered pattern of growth. Using zinc, a metallothionein inducer, we observed a slight increase in the growth rate of some transfectants, which can be measured by thymidine incorporation. However, the relative inefficiency of immortalization we observed suggests that either a high level of myc expression or participation of other genes is required for establishment in culture. Under our experimental conditions, we could not detect a transforming activity for the human myc gene and none of our myc-containing cell lines was tumorigenic in nude mice.

Influence of myc overexpression on the phenotypic properties of Chinese hamster lung cells resistant to antitumor agents

In the Chinese hamster lung fibroblast cell line DC-3F, the development of resistance to different drugs, through several mechanisms like MDR expression or alteration of the DNA topoisomerase II activity, has been shown to be associated with a decreased tumorigenicity. Multiple studies have shown that the myc oncogene, in cooperation with ras, plays a major role in the oncogenic transformation of fibroblasts. As an approach to a better understanding of the relationship between the different phenotypic traits, we analyzed the expression of myc and ras oncogenes in the drug-sensitive DC-3F cells and in variants resistant to 9-hydroxyellipticine (9-OH-E) (DNA topoisomerase II alteration) or to actinomycin D (AD) (multidrug (MDR) expression). Southern and Northern blot analyses revealed about a 10-fold amplification and a 20-fold overexpression of the c-myc gene in the DC-3F cells as compared to the normal lung fibroblasts. Both amplification and overexpression are markedly decreased in the two resistant variants, ras gene copy number and expression were found to be identical in all cell types. In order to analyze the contribution of the decreased myc expression on the different phenotypic traits, the DC-3F/9-OH-E cells were transfected with the plasmid pSV-c-myc, and six clones expressing high amounts of the transfected myc were isolated and characterized. Morphological and caryological alterations, as well as an increased cloning efficiency in soft agar, indicated that the myc gene product was made in these cells. However, the tumorigenicity of the sensitive parental cells was not restored, thus showing that the decreased myc expression alone does not account for the loss of tumorigenicity in the resistant cells. 9-OH-E resistance was not modified in the transfected cells, while the cross-resistance of these cells to MDR-sensitive drugs, such as vincristine, actinomycin D, and taxol, was reversed roughly in proportion of the expression of the transfected myc.

Correlation between an HLA-DQα length polymorphism of messenger RNA and serologically defined specificities (DQwl, DRw53, DR3 + 5)

AbstractmRNAs for the two chains of the HLA-DQ molecule were analyzed, in particular the DQα mRNA whose polymorphism had previously been suggested (Schenning et al. 1984). Northern blot transfers of the mRNA of 12 LCLs and of B lymphocytes from a healthy donor were carried out. We report that a length polymorphism of DQα mRNA exists, and we show that it can be correlated with serologically defined specificities (DQwl, DRw53, DR3 + 5). This correlation could be explained by a linkage disequilibrium, as these specificities are considered to be different from those carried by the DQ molecule (except for the DQw1 specificity).

Induction of pgp3 expression and reversion of the multidrug resistance phenotype in 9-OH-ellipticine-resistant Chinese hamster lung fibroblasts transfected with the MYC oncogene

Chinese hamster lung cells resistant to the DNA topoisomerase II inhibitor 9-OH-ellipticine (DC-3F/9-OH-E) are cross resistant to various drugs through the expression of the MDR phenotype. The myc oncogene was approximately 10-fold amplified and 20-fold overexpressed in parental DC-3F cells as compared with DC-3F/9-HO-E cells. Transfection of the resistant cells with a mouse c-myc gene did not alter the resistance to topoisomerase II inhibitors and, in cells with a low multidrug (MDR) expression, reversed this phenotype. Northern and Western blot analyses revealed an increased expression of pgp1 in the DC-3F/9-OH-E cells, which was not modified in the myc-transfected clones. However, myc expression in these clones resulted in an increased expression of pgp3, roughly in proportion to the level of myc expression. Transfection of the DC-3F/9-OH-E cells with the human MDR3 gene, homologous to pgp3, also resulted in the reversion of the MDR phenotype. These results show that (1) expression of the transfected myc gene positively regulates pgp3 expression but has no effect on pgp1; (2) when observed, reversion of the MDR phenotype is proportional to the levels of myc and pgp3 expression; and (3) this reversion, resulting from pgp3 expression, is associated with a decreased functional activity of the pgp1 protein and might require an appropriate balance of pgp1 and pgp3 expression.