A dynamic regulation of GDNF-family receptors correlates with a specific trophic dependency of cranial motor neuron subpopulations during development.

Glial cell line-derived neurotrophic factor (GDNF) family ligands promote the survival of developing motor neurons in vivo and in vitro. However, not all neurons survive with any single ligand in culture and GDNF null mutant mice display only a partial motor neuron loss. An interesting possibility is that subpopulations of motor neurons based on their function and/or their myotopic organization require distinct members of GDNF family ligands. Because responsiveness to the different ligands depends on the expression of their cognate ligand-binding receptor we have herein addressed this issue by examining the expression of GDNF-family receptors (gfr) during development and in the adult in cranial motor nuclei subpopulations. We have furthermore examined the in vivo role of GDNF for cranial motor neuron subpopulations. The shared ret receptor was expressed in all somatic, branchial and visceral cranial embryonic motor nuclei examined, showing that they are all competent to respond to GDNF family ligands during development. At early stages of development both the GDNF receptor, gfralpha1, and the neurturin (NTN) receptor, gfralpha2, were expressed in the oculomotor, facial and spinal accessory, and only gfralpha1 in the trochlear, superior salivatory, trigeminal, hypoglossal and weakly in the dorsal motor nucleus of the vagus and the ambiguous nucleus. The abducens nucleus was negative for both gfralpha1 and gfralpha2. The artemin (ART) receptor, gfralpha3, was expressed only in the superior salivatory nucleus. A motor neuron subnuclei-specific expression of gfralpha1 and gfralpha2 was seen in the facial and trigeminal nuclei which corresponded to their dependence on GDNF in null mutant mice. We found that the expression was dynamic in these nuclei, which may reflect developmental changes in their trophic factor dependency. Analysis of GDNF null mutant mice revealed that the dynamic receptor expression is regulated by the ligand in vivo, indicating that the attainment of changes in dependency could be ligand induced. Our results indicate that specific GDNF family ligands support selective muscle-motor neuron circuits during development.

The homeodomain transcription factors Islet 1 and HB9 are expressed in adult alpha and gamma motoneurons identified by selective retrograde tracing.

To study gene expression in differentiated adult motoneuron subtypes, we used fluorescent dextrans for both anterograde and retrograde axonal tracing in adult rat and mouse. Application of these dyes to the cut distal and proximal ends of small extramuscular nerve branches revealed both the peripheral ramifications and the cell bodies of subsets of motoneurons. We show that the soleus muscle is innervated by two nerve branches, one of which contains gamma motor and sensory axons but no alpha motor axons. By retrograde tracing of this branch, we selectively labelled gamma motoneurons. In adult rat, the nerves innervating the soleus and extensor digitorum longus muscles contain almost exclusively axons innervating slow (type I) and fast (type 2) muscle fibres, respectively. We selectively labelled slow and fast type motoneurons by retrograde tracing of these nerves. With immunocytochemistry we show that adult motoneurons express several homeodomain genes that are associated with motoneuron differentiation during early embryonic development. Combining selective retrograde labelling with immunocytochemistry we compared the expression patterns in alpha and gamma motoneurons. The homeodomain transcription factors Islet 1 and HB9 were expressed in slow and fast alpha motoneurons and in soleus gamma motoneurons. Motoneurons in each population varied in their intensity of the immunostaining, but no factor or combination of factors was unique to any one population.

Putative structure of the FGF6 gene product and role of the signal peptide.

The human FGF6 gene is an oncogene related by sequence similarities to the fibroblast growth factor (FGF) gene family, which encodes mitogenic peptides implicated in various physiological processes including angiogenesis, morphogenesis, tissue regeneration and survival and oncogenesis. Nucleotide sequence analysis of the FGF6 gene and of cDNA clones revealed an open reading frame able to code for a protein of 208 residues. The FGF6 protein shares 32-70% residues with the other members of the family within the C-terminal two-thirds of the molecule. In vitro, three in-frame ATG codons are able to initiate the translation of three peptides of 175, 198 and 208 residues. These three peptides differ at their amino termini with respect to the relative position of a hydrophobic leader peptide, which extends from residues 16 to 40, and is therefore absent from the shorter (175 amino acids) form. In-vitro analysis indicates that this signal peptide is able to drive the FGF6 protein through the endoplasmic reticulum, where it becomes glycosylated. The presence of this signal peptide sequence appears essential for the in vivo transforming capacity of the FGF6 gene.

Restriction and complexity of Mcf2 proto-oncogene expression.

MCF2/DBL is an X-linked proto-oncogene encoding a protein with a yet undetermined function. It can be activated in vitro by loss of 5' sequences in NIH3T3 bioassays; in vivo, deletion of the gene has been found in some hemophilia B patients. PCR analysis of its expression in mouse tissues shows a restriction to the gonads and tissues of neuroectodermal origin. It also identifies an exon encoding 42 amino acids that is alternatively spliced in murine, but not human testis.

Localization of the HST/FGFK gene with regard to 11q13 chromosomal breakpoint and fragile site.

The HST/FGFK gene, a member of the fibroblast growth factor gene family and a protooncogene, is localized on chromosomal band 11q13. Genes in this region are frequently involved in hematopoietic and solid tumors. Here we show that the HST gene lies telomeric to the BCL1 gene, the t(11;14)(q13;q32) breakpoint, and the FRA11A rare fragile site.

Structure, chromosome mapping and expression of the murine Fgf-6 gene.

The sixth member of the fibroblast growth factor gene family was cloned and analysed in the mouse. It is composed of three coding exons and encodes a putative growth protein of 198 amino acids, possessing a potential signal peptide, and presenting 79% and 93.5% sequence similarity with the mouse Hst/K-fgf and human FGF-6 genes products, respectively. The murine Fgf-6 gene is located in a region distinct from the Int-41 locus and belongs to a linkage group conserved between chromosome 12 in man and chromosome 6 in mouse. It presents an intrinsic oncogenic capacity since it is able to transform cultured fibroblasts. Fgf-6 mRNA levels are developmentally regulated with a peak of expression in the developing fetus at day 15.5 of gestation, moderate levels during late gestation and in the neonate. In the adult, Fgf-6 mRNA can be detected in testis, heart and skeletal muscle.

[Proto-oncogenes activation in human solid tumors]. / Activation de proto-oncogènes dans les tumeurs solides humaines.

Genetic alterations in solid human tumors occur in several groups of genes, one of which contains the proto-oncogenes. These genes are involved in the control of cell proliferation and become activated in oncogenes by various mechanisms. These activations are reviewed herein.

Amplification of FGF-related genes in human tumors: possible involvement of HST in breast carcinomas.

In order to document a possible involvement of structural alterations of FGF (Fibroblast Growth Factor)-like genes in human oncogenesis, we have screened a large series of human tumors for amplification of five FGF-related genes (Basic-FGF, INT2, HST, FGF5 and FGF6). None of 37 hematopoietic neoplasms, one out of 13 melanomas (8%), three out of 43 bladder tumors (7%) and 41 out of 238 breast carcinomas (17%) contained amplified FGF-related sequences, namely HST and INT2. Only these two genes, both located on band q13 of chromosome 11 have been found amplified. In all cases they were co-amplified and in only one instance did amplification extend to the ETS1 locus at position 11q23. INT2 and HST RNA could be evidenced by RNA/RNA in situ hybridization in breast carcinomas. Our results indicate a correlation between RNA expression and gene amplification in the case of HST but not of INT2. Although evaluation of the clinical significance of HST amplification and expression must await long-term follow-up of the patients, we suggest that HST gene product could play a role in development and/or progression of human breast cancer.

Characterization of the HST-related FGF.6 gene, a new member of the fibroblast growth factor gene family.

By screening a mouse cosmid library with a human HST probe under reduced conditions of stringency, we isolated several positive clones. One of them was identified as a new member of the fibroblast growth factor gene family, and called FGF.6. The human FGF.6 gene was subsequently isolated and sequenced. The deduced amino-acid sequence exhibited 70% identity with the HST gene product over the C-terminal two-thirds of the putative protein. FGF.6 was mapped to chromosome 12 at band p13 by in situ hybridization. The cloned normal human gene was able to transform mouse NIH3T3 fibroblasts using both focus- and tumorigenicity-assays.

The FGF-related oncogenes hst and int.2, and the bcl.1 locus are contained within one megabase in band q13 of chromosome 11, while the fgf.5 oncogene maps to 4q21.

Prompted by the observed co-amplification at the DNA level of the int.2 and hst fibroblast growth factor-related oncogenes in some tumor cells, we have investigated the precise localization of these two loci known to reside in band q13 of chromosome 11. We demonstrate by pulsed field gel analysis that these two genes are separated by only 40 kb, locate three HTF islands in their neighbourhood, and show that the bcl.1 locus (translocation breakpoint in B-cell proliferative malignancies) is not more than 1050 kb away. We also show that the fgf.5 gene which belongs to the same family is not part of this cluster and is located in band q21 of chromosome 4.

Relationship between cellular adhesiveness and metastatic activity in polyomavirus-transformed FR3T3 rat cell lines.

A series of polyomavirus-transformed rat cells with varying tumorigenic potential were tested for biophysical parameters possibly related to metastatic properties: adhesive capacity and strength of adhesion to different substrates (laminin, fibronectin and albumin), cell deformability and spreading. Two groups of cell lines were defined according to their higher or lower adhesive capacity. Adhesivity did not appear to be related to cell deformability and spreading. A weak correlation was suggested between low adhesivity and high metastatic potential. A selection method was devised to separate cell samples into 3 subpopulations with different adhesive strength. Two cell lines, originally different, were chosen for this study: Py-tsa A25 cells were less adherent and highly metastatic, and Py-WTA2 cells were more adherent and less metastatic. After s.c. inoculation into syngeneic Fisher rats, the 3 selected subpopulations of the 2 cell lines induced pulmonary nodules to varying degrees, but only the less adherent ones were able to induce visceral metastasis located in stomach and intestine. In this case, animal survival time was 30% lower than for the highly adherent selected cells. After 10 culture passages, the same subpopulations were able to metastasize only in the lungs. However, when the selection procedure was repeated, the less adherent cells were again able to yield visceral nodules. Tumorigenicity remained unchanged in all cases. Study of cell dissemination and arrest in vivo showed a rapid targeting of labelled tumor cells toward lungs and stomach 5 hr after intradermal injection, where they remained up to 72 hr. More adherent cells displayed delayed localization after injection (24 hr) and radioactivity decreased more rapidly.

Chromosomal localization of the hst oncogene and its co-amplification with the int.2 oncogene in a human melanoma.

In this report we described the linkage between two oncogenes of the fibroblast growth factor family. Using in situ hybridization to human metaphase chromosomes we mapped the hst gene to chromosome 11 at band q13. This is also the location of the int.2 gene. Furthermore, the two genes are co-amplified in a human melanoma, raising the possibility that amplification in human tumors may be a mechanism of activation of genes of the FGF family.

Detection of a low frequency of activated ras genes in human melanomas using a tumorigenicity assay.

We have used an assay combining DNA-mediated gene transfer and tumorigenicity in Swiss athymic mice to look for activated ras genes in solid human sporadic melanomas. This assay can detect ras oncogenes mutated at codons 12, 13, or 61. We examined a panel of 13 independent surgical specimens of primary tumors and metastases. No H- or K-ras oncogenes were detected; an N-ras oncogene, mutated at codon 61, was identified in one of the 13 samples. No N-ras genes mutated at codon 13 were detected. Thus, the tumorigenicity assay detects a low frequency of ras gene activation in melanomas.

Detection of transforming sequences in human melanomas and leukemias.

DNA extracted from fresh solid human melanoma tumors and untreated acute myeloid leukemic cells was used in two assays designed to detect oncogenes, based on the transfection of murine NIH 3T3 fibroblasts followed by selection of transformed cells in low serum concentration or induction of tumors in athymic mice. Ras and non-ras oncogenes were detected.

[Activated oncogenes in human tumors]. / Les oncogènes ras activés dans les tumeurs humaines.

ras oncogenes are cellular genes altered by point mutation in 10 to 30% of human tumors. Under this mutated form they play a role in the malignant process, probably in association with other oncogenes. The different ras genes identified in human cancers, the point mutations that activate the ras genes and the properties of the ras proteins are described.

Polyomavirus-transformed FR 3T3 rat cells are able to form metastases in syngeneic rats.

A series of polyomavirus-transformed FR 3T3 rat cell lines were tested for their tumorigenic and metastatic properties after subcutaneous inoculation of syngeneic Fisher rats. All of them grew into tumors, which appeared with variable latency periods; the TD50 varied from cell line to cell line. Eight of the 18 transformants that were inoculated gave rise to metastases, always localized in the lung. The capacity to form metastases, though at a low frequency, was also conferred on FR 3T3 cells upon transformation with a recombinant plasmid encoding only the middle-T protein. Fibroblast-like cells were predominantly observed upon histological examination of the metastases. Culture cell lines were derived from independent tumors and metastases induced by two transformants with low and high metastatic potentials, respectively. Metastasis-derived cell lines exhibited metastatic potentials similar to those of the respective original transformants. All the tumor- and metastasis-derived cell lines synthesized the same early viral polypeptides as the respective original transformants; in contrast, the viral DNA integrations evolved during tumor and metastasis formation.

The transformed phenotype in culture and tumorigenicity of Fischer rat fibroblast cells (FR3T3) transformed with bovine papilloma virus type 1.

Unlike cell lines transformed with Polyomaviruses, transformants derived by focus formation or colony formation in agarose medium after transfer into rat fibroblast cells (FR3T3 line) of Bovine Papilloma Type 1 (BPV1) DNA were consistently observed to grow poorly in suspension and to remain highly serum dependent for growth in culture. These cells did not produce detectable amounts of plasminogen activator, and kept the flat morphology and organized cytoskeleton characteristic of the normal fibroblast. However, they induced in syngeneic animals the development of tumors with a greater invasive potential than tumors induced by Polyomaviruses. By contrast with the original transformants, cells recovered from the tumors grew efficiently in suspension and produced high levels of plasminogen activator. They still had, however, extended cytoskeletal structures and remained completely dependent on high serum concentrations for growth in culture. The stepwise transformation process induced by BPV1 thus appears strikingly different from that previously observed with polyoma and SV40 viruses. The observed changes in transformation phenotype between transformed line and tumor cells do not correlate with any important modification of the number of autonomous copies of the viral genome, nor with any rearrangement of viral sequences detectable at the level of the blot analysis.

Cell lines derived from tumors induced in syngeneic rats by FR 3T3 SV40 transformants no longer synthesize the early viral proteins.

Several SV40-transformed FR 3T3 rat cell lines formed tumors upon inoculation to syngeneic immunocompetent Fisher rats. These tumors, which appeared only after a long latency period, showed a fast rate of growth. Tumor-derived (TD) cell lines were established in culture from several tumors induced by independent transformants, and their properties were analyzed. Though TD cells were highly tumorigenic, their level of transformation in culture was similar to that of the original transformants. They did not synthesize detectable amounts of the two early viral gene products, the large-T and small-T polypeptides. However, the transformation-associated cellular p53 protein was detected in all of them by [35S]methionine labeling and immune precipitation with monoclonal antibodies directed against the mouse p53. Growth in the animal apparently counterselected the cells expressing the early viral proteins, and hence, possibly, the tumor-specific transplantation antigen. This selection was mediated at least in part by the T-cell immune response, as the tumors induced by the same transformants in nu/nu mice still expressed the nuclear T-antigen. Absence of expression of the early viral region was frequently correlated with the loss of the integrated SV40 DNA. Some tumors, however, still contained early viral DNA sequences, which were, in even fewer cases, transcribed into RNA. These results altogether suggest that tumor formation by the FR 3T3-SV40 transformed cells in immunocompetent rats requires two events, the selection for the acquisition of a high tumorigenic potential, and against the expression of the early viral genes. Only the first of these two events was observed upon tumor formation in nude mice.