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Vascular endothelial growth factor (VEGF) binding to the kinase domain receptor (KDR/FLK1 or VEGFR-2) mediates vascularization and tumor-induced angiogenesis. Since there is evidence that KDR plays an important role in tumor angiogenesis, we sought to identify peptides able to block the VEGF-KDR interaction. A phage epitope library was screened by affinity for membrane-expressed KDR or for an anti-VEGF neutralizing monoclonal antibody. Both strategies led to the isolation of peptides binding KDR specifically, but those isolated by KDR binding tended to display lower reactivities. Of the synthetic peptides corresponding to selected clones tested to determine their inhibitory activity, ATWLPPR completely abolished VEGF binding to cell-displayed KDR. In vitro, this effect led to the inhibition of the VEGF-mediated proliferation of human vascular endothelial cells, in a dose-dependent and endothelial cell type-specific manner. Moreover, in vivo, ATWLPPR totally abolished VEGF-induced angiogenesis in a rabbit corneal model. Taken together, these data demonstrate that ATWLPPR is an effective antagonist of VEGF binding, and suggest that this peptide may be a potent inhibitor of tumor angiogenesis and metastasis.

Sodium phenylacetate (NaPa), a physiological product of phenylalanine metabolism, present in micromolar concentrations in human plasma, has been shown to induce in vivo and in vitro cytostatic antiproliferative effects at millimolar concentrations. Cadherin molecules are powerful invasion suppressor molecules and the reduction of E-cadherin expression plays an important role in the invasion and metastasis of human breast cancer. In this study, we demonstrated, on one hand, that NaPa stimulated aggregation by increasing the expression of E-cadherin at the surface of breast cancer MCF-7ras cells transformed by Ha-ras oncogene and inhibited its expression in MCF-7 cells. We demonstrated that NaPa increased the formation of MCF-7ras cell aggregates and did not alter the formation of MCF-7 cell aggregates. By Northern blot, we demonstrated that the E-cadherin expression was not regulated at the transcriptional level. On the other hand, we analyzed the cell cycle of these 2 cell lines after NaPa treatment and showed that NaPa induced arrest at the G1/S phase in both MCF-7 and MCF-7ras cells. bFGF increased the growth of MCF-7 cells, but inhibited MCF-7ras cell proliferation. NaPa treatment suppressed the stimulation of MCF-7 cell proliferation and increased MCF-7ras cell growth inhibition. We have demonstrated a new target of NaPa action in blocking the cell cycle of tumor cells in G0/G1. We suggest that the anti-proliferative effect of NaPa associated to the restoration of the cadherin function in human mammary carcinoma cells indicates that NaPa could be a novel therapeutic agent in breast cancer.

The comparative tumorigenicity in rats and nude mice of cell lines derived from FR3T3 and transformed by either c-jun, ras, SV40 lt, or bovine papilloma virus type 1 (BPV1) oncogenes was investigated. c-Jun-transformed cells were as tumorigenic and metastatic as Ras-transformed cells. Latencies were short, and numerous pulmonary metastases were observed in all injected animals. In contrast, tumors induced by s.c. injection of SV40-transformed cells developed slower, and none of the animals who received injections i.v. presented with metastases. BPV1-transformed cells had an intermediate tumorigenic and metastatic activity. Microvessels present in the different tumors were revealed by immunostaining with Griffonia (Bandeiraea) Simplicifolia lectin 1. Tumors obtained with c-Jun-transformed cells exhibited more neovascularization than those induced by the other oncogenes. By comparison to FR3T3 cells or SV40- or BPV1-transformed cells, c-Jun-transformed fibroblasts repress the antiangiogenic thrombospondin-1 and SPARC genes, whereas we found that they express higher levels of gene expression of the angiogenic vascular endothelial growth factor. Finally, as compared with cells before passage in animals, thrombospondin-1, SPARC, and VEGF gene expression was also deregulated in cell lines isolated from primary tumors induced by BPV1-transformants. Our results indicate that the high transforming potential of c-Jun, evidenced as soon as transformation is established in vitro, correlates with deregulation of gene expression of both angiogenic and antiangiogenic factors leading to rapid neovascularization of tumors.

Sodium Phenylacetate (NaPq) has been shown to suppress tumor growth and promote differentiation in experimental models. Thus, we have previously shown an inhibition of MCF-7ras cell proliferation by NaPa both in vitro and in vivo on xenographed tumors. In order to study the action of NaPa on the synthesis of paracrine or autocrine growth factors, conditioned media were prepared from breast pretumoral HBL100 cells, tumoral MCF-7 and MCF-7ras cells in the presence of NaPa. Growth factor activities of these media were tested on Balb c/3T3 fibroblasts and on the above breast tumor cells. Conditioned media from the 3 cell types contained different mitogenic activities when tested on the same cell lines. NaPa treatment for 24 hours inhibited differentially and dose-dependently the mitogenic activity of conditioned media. Inhibitions of HBL100 and MCF-7 cell proliferation by MCF-7ras medium conditioned with 20 mM NaPa reached 75% and 48% respectively. In contrast, NaPa treated MCF-7 conditioned medium decreased HBL100 and MCF-7ras proliferation by 49% and 72%, respectively, at the same NaPa concentration. The efficiency of NaPa inhibition reached an optimum as soon as one day after treatment. Among growth factors secreted by MCF-7 and MCF-7ras, TGF beta synthesis is inhibited and stimulated in MCF-7 and MCF-7ras cells respectively after NaPa treatment. We showed that NaPa modifies the synthesis of growth factors secreted by MCF-7 and MCF-7ras tumor cells leading to cell proliferation inhibitions. The synthesis of these previously identified factors was more involved in MCF-7 cells than fibroblast cell proliferation. In vitro and in vivo NaPa inhibition of MCF-7ras cells which secreted higher levels of these growth factors could be explained by this mechanism of action.

Mouse adrenal medulla was transplanted to mouse brain for morphological and morphometric examination of the nerve endings abutting on the surface of the grafted adrenal chromaffin cells. To determine the types of these endings, they were treated with antibodies specific for phenylethanolamine N-methyltransferase (PNMT), choline acetyltransferase (ChAT) and acetylcholinesterase (AChE). Three types of vesicles were found in nerve fibers and endings: the first contained small clear synaptic vesicles 30-50 nm in diameter, the second was mixed with large granules with moderately electron-dense cores 80-100 nm in diameter, and the third exhibited small electron-dense cored vesicles 50 nm in diameter. The two first types occurred in nerve endings of normal and grafted medulla, but the third was only seen in the grafts. Grafted chromaffin cells carried two morphologically distinct types of synapse: small with a diameter of 1-2 microns, and large, as in normal adrenal medulla. The first type predominated after transplantation. In normal medulla, the number of synapses calculated per grafted chromaffin cells was about 4.5 for cells containing epinephrine (E) and 5.8 for those containing norepinephrine (NE), and in grafted medulla, 4 per cells. After grafting, nerve endings were labeled to ChAT, AChE and neuron-specific enolase (NSE), but only a few nerve fibers were immunoreactive to PNMT. The presence of NSE in nerve endings on the grafted cells, a marker of the glycolytic activity in neurons, suggests the formation of de novo functional synaptic connections.

The graft of chromaffin adrenaline-containing (A) cells of rabbit adrenal medulla implanted to mouse brain and treated with NGF contains more survived cells 1 month after grafting than adrenal medulla alone. The cells developed either an intermediate (e.g. chromaffin cell and neuron) or a neuron-like phenotypes accompanied with a decrease in an immunoreactivity for PNMT (phenyletanolamine-N-methyltransferase). A gap junctions and attached plaques were found between grafted cells. The grafts received a synaptic input. The NGF influence on the fate of chromaffin A-containing cells is discussed.

The expression of two oligodendroglial markers, galactocerebroside (GC) and myelin basic protein (MBP), was studied in brain cell cultures (BCC) from 14-day-old mouse embryos by immunocytochemical methods. The presence of neurons and astrocytes was also investigated. Results show that oligodendrocytes simultaneously express both GC and MBP already at 7 days in vitro. These cultures are rich in neurons, and the astrocyte layer is also well represented. A comparison is made between these data and those previously obtained by the use of newborn mouse brain cell cultures, which are very poor in neurons. The differentiation of oligodendrocytes, as reflected in the expression of MBP, is accelerated in embryonic mouse BCC when compared to neonatal mouse BCC. We therefore speculate that neurons are involved in the enhancement of the ability of oligodendrocytes to express myelin related components in culture.

The polysaccharides were studied in an electroreceptor organ, the tuberous organ of Gnathonemus petersii (Mormyridae). Histochemical methods (P.A.S., alcian blue, toluidine blue and iron colloidal reactions) allowed us to demonstrate the existence of glycogen in the sensory cytoplasm, and P.A.S. positive polysaccharides in the sensory cavity. The polysaccharides were shown to be amylase proof; they display an acidity due to the existence of sulphated radicals. The histochemical study was completed by a cytochemical analysis: a treatment with thiocarbohydrazide (TCH) according to the Thiery's method. This method allowed us to estimate the glycogen concentration, its localization, and relationship with cellular organites within the sensory cytoplasm, as well as to differentiate the highly glycogenous type II cells of the platform from the other accessory cells (Derbin and Szabo, 1968). After a treatment for 20 hours with TCH, silver stained grains were visible on the polysaccharide filaments of the sensory chamber, between the microvilli and the vacuoles of the epidermal cells lining to the sensory cavity. Silver grains coated the outer surface of the microvilli. Such polysaccharides were not identical to the filamentous polysaccharides of the cavity. In order to determine the cytochemical localization of the polysaccharide acid groups, sections were stained with iron salts. The colloidal iron constitutes a deposit opaque to electrons and located both on the filamentous polysaccharides of the sensory cavity and in the vacuoles of the epidermal cells, indicating that only these filamentous polysaccharides display acid radicals.

Mechanically dissociated brain cells of 14 and 18-day-old mouse embryos and of mouse neonates were cultured for 3 weeks. Neurons, oligodendrocytes and astrocytes were identified at the 7th, 14th and 21st day in vitro by staining the cultures using the indirect immunoperoxidase technique with antisera directed against neuron specific enolase, galactocerebroside, myelin basic protein and glial fibrillary acidic protein. The number of neurons and oligodendrocytes was higher in embryonic cultures than in neonate cultures. The expression of some antigens was also different in the two types of culture. Our results indicate that the development of brain cells in mechanically dissociated brain cell cultures depends on the age of the animal at the time of plating.

The penetration of the CVS strain of rabies virus and its avirulent derivative AvO1 into peripheral neurons was investigated after intramuscular inoculation into the forelimbs of adult mice. It was found that CVS directly penetrates both the sensitive and motor routes with equal efficiency, without prior multiplication in muscle cells. Infected neurons became detectable 18 h after infection. The second cycle of infection occurred within 2 days, and at day 3 there was a massive invasion of the spinal cord and sensory ganglia. In sensory ganglia, where it was possible to identify cell outlines, it was evident that the infection did not proceed directly from cell body to cell body. The avirulent strain AvO1 penetrated motor and sensory neurons with the same efficiency as CVS. Restriction of viral propagation was observed from the second and third cycles onwards. No further development of the infection could be seen after day 3, and by that time the lysis of primarily infected neurons seemed to occur.

Antibodies against Z-DNA bind to fixed metaphase chromosomes of man and Cebus albifrons (Platyrrhini, Primate). By indirect immunofluorescence and indirect immunoperoxidase techniques, a heavy staining is detected in some segments of chromosomes of C. albifrons. These segments correspond to R-band-positive heterochromatin, which has a high G + C-base content. Euchromatin of human and Cebus chromosomes show a weak and heterogeneous staining that consistently reproduces an R- and T-banding pattern in both species. Because chromosome homologies previously were demonstrated between these distantly related species by chromosome banding, our results suggest that Z-DNA has been conserved during the course of primate evolution.

Antisera and antibodies against Z-DNA were used on metaphasic fixed chromosomes of a mammal, Gerbillus nigeriae (Gerbillidae, Rodentia). By indirect immunofluorescence and indirect immunoperoxidase labelling a heavy staining was detected in a fraction of the R-band positive heterochromatic segments, which are presumed to be rich in G-C base pairs. A weak and non homogeneous staining was also observed on euchromatic segments. The presence of a left-handed Z-DNA in mammalian chromosomes is discussed.