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Two clones of the L5 myoblast line (M6 and the fusion-defective M12) were examined for the expression of myogenin, one of the regulatory genes involved in the regulation of differentiation to myofibers after treatment with 3-deazaadenosine, a metabolic inhibitor of methyl transfer reactions. Cultures treated with 3-deazaadenosine showed, using Northern blot hybridization, a conspicuous increase in myogenin expression, which in clone M6 correlated to the extent of cell differentiation under fusing conditions but was evident also in growth medium, although the drug was unable to start the myogenic program. We also tested the extent of total DNA methylation to verify whether the activation of the regulatory cascade could be correlated to the decrease of the overall number of 5-methylcytosines present in the genome. The results show that the loss of 5-methylcytosine from newly synthesized DNA, but not from preexisting DNA, is evident in fusing conditions and enhanced by 3-deazaadenosine. It appears that there is a positive correlation between the passive demethylation of newly synthesized DNA, the activation of the myogenin gene by demethylation, and the differentiation of myoblasts. However, in fusing conditions, the defective clone M12, although it is able to express myogenin and its DNA is hypomethylated, fuses only in the presence of 3-deazaadenosine, suggesting some alternative way of induction.

Retinoic acid (RA) controls the differentiation of a variety of cell types, although its role in influencing T cell development and the mechanisms potentially involved have not been thoroughly investigated. To study the ability of RA to modulate T cell development, we established a thymic stromal cell line (TC-1S) that supports the phenotypic maturation of CD4-8- double negative (DN) or CD3-4-8- triple negative (TN) thymocyte precursors. Cocultures of either DN or TN thymocytes on a monolayer of TC-1S cells resulted in the appearance of thymocytes with a more mature phenotype (CD4+8+ double positive, CD4+ or CD8+ single positive, and CD3(low) cells). Double negative T cell contact with TC-1S cells also increased the production of fibronectin (FN) by the thymic stroma and the expression of the VLA-4 FN receptor on the DN cells. Ab-mediated inhibition of the interaction between FN and its receptors significantly reduced the level of induced T cell maturation. Addition of RA either to TC-1S cells alone or to the coculture with DN cells decreased stromal cell FN expression, antagonized DN cell-induced increase in stromal cell FN production and significantly inhibited in vitro thymocyte maturation. The effects of RA were likely mediated by RA acid receptors alpha and gamma expressed both in DN thymocytes and TC-1S cells. Together these data suggest that FN/VLA-4 interaction may be an important component of stromal cell-dependent thymocyte phenotypic differentiation and that this interaction can be one of the targets for the influence of RA in T cell development.

The effects of an antisense oligodeoxynucleotide to codons 2-7 of the oncogene MYCN on the human neuroblastoma cell line LAN-5 were studied. Treated cells showed a decreased MYCN protein expression and synthesis by immunoperoxidase staining and immunoprecipitation. At the same time, the replication rate was inhibited, and the phenotype was modified toward a more differentiated type. Our data suggest the involvement of oncogene MYCN in both proliferative and differentiative processes.

Myosins and myosin light chain kinases have been isolated from a cloned line of myoblasts (L5/A10) as this cell line undergoes differentiation toward adult muscle. At least three myosin isozymes were obtained during this developmental process. Initially a nonmuscle type of myosin was found in the myoblasts. The molecular weights of the myoblast light chains were 20 000 and 15 000. Myosin isolated from early myotubes had light chains with molecular weights of 20 000 and 19 500. Myosin isolated from myotubes which contained sarcomeres had light chains with molecular weights of 23 000, 18 500, and 16 000. This last myosin was similar in light chain complement to adult rat thigh muscle. Two forms of the myosin light chain kinase activity were detected: a calcium-independent kinase in the myoblasts and a calcium-dependent kinase in the myotubes with sarcomeres. No myosin light chain kinase activity was detected in the early myotubes.

Differentiation of human neuroblastoma (NB) was studied in vitro with five NB cell lines treated with dibutyryl cyclic adenosinemonophosphate and retinoic acid. Although the above agents induced different responses in the various cell lines, three overall morphologic phenotypes emerged: a neuronal, characterized by cell processes and neurosecretory granules, a flat cell without pigment, which displayed basal lamina pertinent to Schwann cells, and a flat pigmented cell which exhibited melanosomes, similarly to melanocytes. The activity of the Schwann cell enzyme cyclic nucleotidyl phosphohydrolase increased considerably in one condition, after induction of a predominantly flat cell phenotype. All studied NB cell lines were capable of synthesizing and expressing the extracellular matrix proteins laminin (LM), fibronectin (FN), and Type IV collagen; but a specific pattern of expression emerged after differentiation, which was proportional to normal tissue equivalents: neuronal--none; melanocytic--FN only; and Schwann cell--large amounts of FN, LM, and Type IV collagen.

HMGI-C and HMGI(Y) are architectural DNA-binding proteins that participate in the conformational regulation of active chromatin. Their pattern of expression in embryonal and adult tissues, the analysis of the "pygmy" phenotype induced by the inactivation of the HMGI-C gene, and their frequent qualitative or quantitative alteration in experimental and human tumors indicate their pivotal role in the control of cell growth, differentiation, and tumorigenesis in several tissues representative of the epithelial, mesenchymal, and hematopoietic lineages. In contrast, very little information is available on their expression and function in neural cells. Here, we investigated the expression of the HMGI(Y) and HMGI-C genes in neuroblastoma (NB), a tumor arising from an alteration of the normal differentiation of neural crest-derived cells and in embryonal and adult adrenal tissue. Although HMGI(Y) is constitutively expressed in the embryonal and adult adrenal gland and in all of the NB cell lines and ex vivo tumors examined, its regulation appears to be associated to growth inhibition and differentiation because we observed that HMGI(Y) expression is reduced by retinoic acid (RA) in several NB cell lines that are induced to differentiate into postmitotic neurons, whereas it is up-regulated by RA in cells that fail to differentiate. Furthermore, the decrease of HMGI(Y) expression observed in RA-induced growth arrest and differentiation is abrogated in cells that have been made insensitive to this drug by NMYC overexpression. In contrast, HMGI-C expression is down-regulated during the development of the adrenal gland, completely absent in the adult individual, and only detectable in a subset of ex vivo NB tumors and in RA-resistant NB cell lines. We provide evidence of a causal link between HMGI-C expression and resistance to the growth arrest induced by RA in NB cell lines because exogenous HMGI-C expression in HMGI-C-negative and RA-sensitive cells is sufficient to convert them into RA-resistant cells. Therefore, we suggest that HMGI-C and HMGI(Y) may participate in growth- and differentiation-related tumor progression events of neuroectodermal derivatives.

Terminal differentiation of myogenic cells has long been known to be positively regulated by insulin-like growth factors (IGFs). Arg8-vasopressin (AVP) has been recently reported to potently induce myogenic differentiation. In the present study, the effects and the mechanisms of action of AVP and IGFs on myogenic cells have been investigated under conditions allowing growth and differentiation of myogenic cells in a simple serum-free medium. Under these conditions, L6 and L5 myogenic cells slowly proliferate and do not undergo differentiation (less than 1% fusion up to 7 days). AVP rapidly (2-3 days) and dose-dependently induces the formation of multinucleated myotubes. Creatine kinase activity and myosin accumulation are strongly up-regulated by AVP. Insulin or IGF-I or IGF-II, at concentrations that cause extensive differentiation in serum-containing medium, induces a modest degree of differentiation in serum-free medium. The simultaneous presence of AVP and of one of the IGFs in the synthetic medium induces maximal differentiation of L6, L5, and satellite cells. The expression of both myogenin and Myf-5 is dramatically stimulated by AVP. Our results indicate that AVP induces a significant level of myogenic differentiation in the absence of other factors. Furthermore, they suggest that to express their full myogenic potential, IGFs require the presence of other factors normally present in serum and fully mimicked by AVP. These studies support the conclusion that terminal myogenic differentiation may depend on the presence of differentiation factors rather than the absence of growth factors.