Francis Berthelot

Effects on the cytoskeleton of a new inducer of the neuroblastoma morphological differentiation

Abstract 1-Methyl cyclohexane carboxylic acid (CCA), a new inducer of neuroblastoma morphological differentiation, was found to stimulate the synthesis of vimentin in neuroblastoma cultures. Synthesis of actin and isotubulins was also modified, thus implying a more general effect on the cytoskeleton. Most of these variations are observed in culture conditions allowing interactions between the cell membrane and a solid substrate.

Complexity of polysomal polyadenylated RNA in mouse whole brain and cortex

Data concerning the eukaryotic messenger sequence complexities broadly differ according to the techniques of measurement. The analysis of hybridization kinetics of cDNA to mRNA template tends to underestimate the complexity as compared to saturation hybrid~ation of single copy DNA. This discrepancy has been considerably emphasized in the case of mouse brain poly~A)RNA. The data range from w 10 000 sequences [ 1 ,l’@J to 100 000 f 121. Causes for this discrepancy are not totally explained, however, by the difference in the technical approaches, since important variations also appear in the results reported by authors using the same technique. For instance, using cDNA, -19 000 sequences were found [2] as compared to 11 600 [ 11. In many cases, these variations may be explained, at least partly, by differences in the experimental protocols or (and) by the use of two different analytical methods (computer analysis or linear plot) for obta~i~g best fit to the data. Independently of the technical approach, and its obvious impact on the quantitative conclusions, a higher sequence complexity has been claimed [2-51 for mouse brain than for other tissues. These authors generally interpret the high level of poly(A)RNA sequence complexity as reflecting the high heterogeneity of cell types constituting the brain. The possibility has not been totally excluded, however, that a neuronal cell per se, perhaps because of its very large potential in estabB~g homotypic and hetero~p~c contacts, would express more genes than cells from any other tissues.

Effects on mitochondrial metabolism of CCA, one inducer of neuroblastoma differentiation

CCA, a potent neuroblastoma differentiation inducer, was shown by oxygraphic measurements to reduce significantly the O2 consumption of whole neuroblastoma cells as of mitochondria purified from neuroblastoma or mouse cortex. The effect of CCA on the respiration was compared to those of oligomycin. Our results suggest that the molecular target of CCA is the matrix F1 catalytic component of the F0F1 mitochondrial ATPase.

Changes in the pattern of poly(A)-containing RNA during terminal differentiation in neuroblastoma cells.

One of the difficulties in analysing the control of genetic expression during the terminal differentiation of somatic cell systems is to decide whether the parameter under study relates to the acquisition of a new phenotype or to more general preparative events, such as the entrance into the post-mitotic stage. In the case of a neuronal system, such as mouse neuroblastoma cells, this situation is complicated by the fact that the developmental stages under comparison often refer to cell populations cultivated in different nutritional or physical conditions. Two stages are often analysed: round immature neuroblasts grown in suspension in the presence of serum or cells exhibiting the properties of mature neurones after being attached to culture dish in the absence of serum. This remark particularly applies to previous observations from the literature in which the synthesis of various enzymes, or of tubulin, have been compared in the course of neuroblastoma differentiation [ 1 -4 ] . It also refers to our previous work on the transcriptional activities of immature and differentiated neuroblastoma [5]. Analysis of the hydrodynamic distribution of cytoplasmic poly(A)-containing RNA species from suspension or monolayer grown cells has revealed interesting differences with the virtual disappearance in the latter kind of cells of the 16 S pulse-labelled fraction and a relative variation in the amplitudes of some other fractions. Interestingly the 15 S fraction, observable after pulse-labelling of the suspension grown cells but not after labelling of the monolayer culture, could however be detected in this latter case by hybridization to [3H]poly(U) when using polysomal poly(A)-containing RNA. This was taken as evidence that morphological differentiation does not involve major alteration of the transcription program, but variations at the level of the relative stability of some cytoplasmic messengers. A question that remained unanswered however was whether change~ observed in the pattern of pulselabelled species did in fact correspond to the morphological events accompanying terminal differentiation or to the modifications in the culture conditions which were experimentally introduced to trigger the developmental program. In particular, the relevance of the transition between the logarithmic growth of suspension cells and the postmitotic stationary phase of monolayer cultures had to be explored. The present study attempts to answer the question. Cytoplasmic poly(A ÷) RNA distribution has been reinvestigated in populations of neuroblastoma attached to culture dish, in a stationary phase, under conditions where no neurite extension occurs. This was achieved by changing the culture conditions in the case of the NIE 115 clone or by using a special neuroblastoma variant, clone NIA 103, which does not undergo morphological differentiation after induction by serum withdrawal [6]. Our results are consistent with the conclusion that there is a causality relationship between the changes in the distribution of poly(A)-containing RNAs previously described [5] and the formation of neurites.

Changes in mitochondrial proteins during neuroblastoma differentiation

The evolution of three major mit-proteins was followed in neuroblastoma cells cultured in different conditions of differentiation. 1 methyl cyclohexane carboxylic acid (CCA) was found to stimulate the synthesis of the three mit-protein markers. This result, compared to the effects of oligomycin, an inhibitor of mitochondrial function, favours the hypothesis that CCA induces in vitro neurogenesis through a general metabolic alteration.

Effects of 1-methyl cyclohexane carboxylic acid (CCA) on cellular energetics in neuroblastoma cells.

Abstract The level of cellular energetics has been estimated in neuroblastoma cells under different culture conditions. The cellular accumulation of isomerase-2-deoxy [ 14 C]D-glucose-6-phosphate was taken as reflecting glucose utilization. A considerably higher amount of radioactivity — 2.5 to 4 times — is found in CCA treated cells, as compared to other types of cultures, corresponding to a higher rate of deoxyglucose penetration and utilization.

Is the induction of neuroblastoma differentiation by CCA mediated by its effects on the electrochemical gradient

Various mitochondrial inhibitors are tested in neuroblastoma cells. Their effects on the mit‐proteins and some cytoskeletal proteins are compared to those of CCA, a differentiation inducer. This comparison favours the hypothesis that the primary effect of CCA induction is an alteration of the electrochemical gradient.

Molecular Approach to the Study of Neural Function and Differentiation

Neurobiology constitutes one of the most challenging aspects of cellular and development biology due to the complexity of the central nervous system and to the diversity of the behavioral patterns among evolved eukaryotic organisms.

Comparative nature of the reactions catalysed by reticulocyte initiation factor IF-M1, and Escherichia coli factor IF2 on Escherichia coli ribosomes

It is well known that initiation of protein synthesis involves fMet tRNA”ff in prokaryotes and Met tRNA”ff in eukaryotes. Although the requirement for the N-formyl substitution of the initiator tRNA is absolute with prokaryotic ribosomes, the reverse is not true. The binding of both Met and fMet tFWAMPt on 80 S ribosomes or on 40 S subunits can be achieved in the presence of the eukaryotic factor IF-M1 [ 1,2] . The reaction with the formylated tRNA is however different from a physiological initiation, because of its lack of GTP requirement. The function of factor IF-M1 thus remains to be elucidated. More precisely, the following questions can be raised. Is IF-M, able to function as a true initiation factor in an E. coli system and, if so, with what efficiency? What is the degree of similarity between IF-M1 and the bacterial factor IF2 which plays a similar role? In a previous study we have already reported on the degree of interchangeability between bacterial and eukaryotic factors with respect to cognate or heterologous ribosomes. The present paper extends part of this study in showing that the E. coli fMet tRNA binding to 70 S ribosomes, directed by IF-MI, leads to occupancy of the same site as that involved in the normal IF? mediated reaction; however, properties of the complex thus formed suggest that the eukaryotic factor cannot be recycled.

Complexity of polysomal poly(A)RNA in different developmental stages of a non-differentiating neuroblastoma clone.

In [ 11, using RNA-DNA molecular hybridizations according to [2-41, we analyzed the sequence complexity and the frequency distribution of the polysomal poIy(A) RNA from NI El 15 neuroblastoma cells at two developmental stages: either as round immature neuroblasts grown in suspension in the presence of serum, or as neurite-bearing cells attached to a culture dish,in the absence of serum. Both mRNA populations exhibited a total complexity corresponding to -7200 average-sized sequences of 1750 nucleotides distributed in 3 abundance classes. These data agree well with those in [5]. All the sequences from differentiated cells were present in the polysomes of undifferentiated cells. Conversely, the mRNA from differentiated cells failed to hybridize with -15% of the cDNA pertaining from cell suspensions. Our interpretation implied that morphological differentiation was accompanied by the disappearance of a small number of messengers corresponding to the intermediate frequency class in the suspension cells. This loss of sequences may be related to the expression of neuronal morphogenesis. However, the possibility that these changes reflected, at least partly, the disappearance of mRNA species involved in cell replication was not ruled out by studies on other systems [4,6]. Here, we analyze the sequence complexities and frequency distributions of polysomal poly(A) RNA from a neuroblastoma variant, clone NlA103, which is unable to undergo neuronal morphogenesis, as expressed by the extension of neurites, under any type of culture conditions assayed. To compare both studies, we used the same technical procedure as for the analysis of NlEl 15 complexities. Furthermore, N 1 A 103 cells have been maintained in the same con-