Glycogen synthase kinase-3beta and the p25 activator of cyclin dependent kinase 5 increase pausing of mitochondria in neurons.

The complex bi-directional axoplasmic transport of mitochondria is essential for proper metabolic functioning of neurons and is controlled by phosphorylation. We have investigated by time-lapse imaging the effects of increased expression of glycogen synthase kinase-3beta (GSK-3beta) and of the p25 activator of cyclin dependent kinase 5 on mitochondria movements in mammalian cortical neurons and in PC12 cells. Both GSK-3beta and p25 increased the stationary behaviour of mitochondria in PC12 and in neurons, decreased their anterograde transport but did not affect the intrinsic velocities of mitochondria. The microtubule-associated tau proteins were more phosphorylated in GSK-3beta and p25 transfected neurons, but ultrastructural observation showed that these cells still contained microtubules and nocodazole treatment further reduced residual mitochondria movements in GSK-3beta or p25 transfected neurons, indicating that microtubule disruption was not the primary cause of increased mitochondrial stationary behaviour in GSK-3beta or p25 transfected neurons. Our results suggest that increased expression of GSK-3beta and p25 acted rather by decreasing the frequency of mitochondrial movements driven by molecular motors and that GSK-3beta and p25 might regulate these transports by controlling the time that mitochondria spend pausing, rather than their velocities.

Molecular characterization of cell substratum attachments in human glial tumors relates to prognostic features.

Glioma cell attachments to substratum play crucial roles in the invasion by glioma cells of normal brain tissue. These attachments are mediated through interactions between extracellular matrix (ECM) components, integrins, focal adhesion-linked molecules, and the actin cytoskeleton. In the present study, we investigate the molecular elements involved in cell substratum attachments in human glial tumors and their potential relationships to prognostic features. We used 10 human glioma cell lines, for which we characterized glial differentiation by means of quantitative RT-PCR for nestin, vimentin, and GFAP mRNA. We quantitatively determined the amounts of laminin, fibronectin, vitronectin, and thrombospondin secreted by these glioma cell lines in vitro, as well as the amount of each of the eight beta integrin subunits and the adhesion complex-related molecules, including talin, vinculin, profilin, zyxin, alpha-actinin, paxillin, and VASP. After quantification of the levels of migration and invasion of these 10 cell lines in vitro and, through grafts into the brains of nude mice, of their biological aggressiveness in vivo, it appeared that the levels of the beta 5 integrin subunit and alpha-actinin were directly related to biological aggressiveness. These experimental data were clinically confirmed because increasing immunohistochemical amounts of the beta 5 integrin subunit and alpha-actinin were directly related to dismal prognoses in the case of astrocytic tumors. In addition, we show that the beta 4 integrin subunit are expressed significantly more in oligodendrogliomas than in astrocytic tumors. A potential role for the beta 8 integrin subunit in glioma cell substratum attachments is also emphasized.

Distinct differences in binding capacity to saccharide epitopes in supratentorial pilocytic astrocytomas, astrocytomas, anaplastic astrocytomas, and glioblastomas.

We monitored the expression of glycan-binding sites on a panel of 10 biotinylated neoglycoconjugates by means of quantitative computer-assisted microscopy to further study the molecular mechanisms in the extensive infiltration of the surrounding brain parenchyma by most astrocytic tumors. Three distinct histological compartments were analyzed for each of the 108 astrocytic tumors (15 pilocytic astrocytomas (WHO grade I), 25 astrocytomas (WHO grade II), 30 anaplastic astrocytomas (WHO grade III), and 38 glioblastomas (WHO grade IV) included in our series. These compartments were tumors (nonperivascular tumor astrocytes), perivascular tumor astrocytes, and blood vessel walls. Clear differences were observed between the pilocytic and the diffuse astrocytic tumors. Furthermore, malignant progression in the latter category was paralleled by a decrease in cells' ability to bind distinct sugar epitopes, especially the D-GalNAc(alpha1-3)-D-GalNAc-beta1-R determinant of the Forssman pentasaccharide in tumors, the alpha-L-fucose in perivascular tumor areas, and the beta-D-glucose in tumor vessel walls. Markedly, the level of binding site expression for alpha-D-mannose decreased in the tumors, the perivascular tumor areas, and the vessel walls. These glycohistochemical results imply the functional relevance of protein-carbohydrate interactions in this tumor system.

Characterization of TNP-470-induced modifications to cell functions in HUVEC and cancer cells.

The aim of the present work is to characterize (both in vitro and in vivo) the influence of TNP-470 on different cell functions involved in angiogenesis and, more particularly, on endothelial cell growth, cell migration and vessel formation. In addition, a possible direct anti-tumor activity was investigated. To this end, we made use in vitro of human umbilical cord endothelial vein (HUVEC) cells and two human cancer cell lines. The TNP-470 effects on the growth of cancer cell lines were compared to those of Taxol (an inhibitor of microtubule depolymerization), a cytotoxic reference which also displays strong antiogenic activity at low (non-toxic) doses. The in vitro effects were characterized on the mouse mammary MXT adenocarcinoma, on which we also characterized the influence of three clinically active anti-tumor compounds (as cytotoxic references). The purpose of this part of the study was to determine the actual TNP-470-related anti-tumor activity and to evaluate the possible toxic side-effects at the doses at which this compound induces tumor growth inhibition. These investigations were completed by analyzing the TNP-470 effects on HUVEC cell motility and in vitro and in vivo vessel formation. The results show that in vitro, TNP-470 inhibited the growth not only of HUVEC, but also of neoplastic cells. Furthermore, TNP-470 clearly inhibited in vitro endothelial cell motility (p<10(-5)). However, it had only a minor effect (p=0.02) on the formation of HUVEC cell networks on Matrigel(R). In vivo, TNP-470 was able to inhibit tumor growth (on the MXT model) at a dose (50 mg/kg) associated with toxic side-effects. Histological examination showed a significant inhibition of vessel formation (p<0.001) at high (toxic) and intermediary (non-toxic) doses (50 and 20 mg/kg). However, we also observed that TNP-470 stimulated lymphocyte proliferation. Thus, care must be taken with the TNP-470 compound in combination with other anti-tumoral agents in order to avoid certain unfortunate clinical complications.

Characterization of biological features and chemosensitivity of a new experimental lung metastasis model originating from the MXT mouse mammary adenocarcinoma.

The present study shows how an original mouse metastatic lung model was established from the MXT mammary adenocarcinoma. This metastatic model was obtained by injecting the C/MET clone into the tail veins of B6D2F1 mice. The C/MET clone corresponds to one of eleven cell clones that were isolated in vitro from the MXT model. Of these 11 clones, only the C/MET leads to lung metastatic tumor development when injected i.v. into mice. Furthermore, the C/MET clone colonizes the lung only. The present data show that the C/MET metastatic model and the MXT parental line are weakly (if reference is made to the P388 leukemia model) sensitive to adriamycin, clyclophosphamide and etoposide. However, under specific experimental conditions, the chemosensitivity of the C/MET model can be significantly increased. The C/MET model therefore appears to be an interesting pharmacological tool to test new investigational agents with anti-tumor potentialities to lung metastases.

Lectin histochemistry, ploidy level, and proliferative activity in rhabdomyosarcoma subtypes.

The glycohistochemical expression of binding sites for eight lectins is characterized in a series of 8 embryonal, 4 alveolar and 4 pleomorphic rhabdomyosarcomas. The correlation between lectin staining and either the proliferation index or the ploidy level was also investigated. The data show that rhabdomyosarcomas exhibit heterogeneous lectin binding expressions. A comparable level of lectin labeling is observed in euploid and aneuploid tumours. In contrast to other neoplasms, lectin staining has proved to be of doubtful value in distinguishing between different RMS subtypes. The data also reveal that a significantly lower level of proliferative activity was observed in the pleomorphic group as compared to the alveolar one.

Influence of dihydrotestosterone, epidermal growth factor, and basic fibroblast growth factor on the cell kinetics of the PC3, DU145, and LNCaP prostatic cancer cell lines: relationship with DNA ploidy level.

The cell kinetics (percentage of cells in the S+G2 phases of the cell cycle) and the DNA ploidy levels (nuclear DNA content) were determined in 108 samples each of the PC3, DU145, and LNCaP prostate cancer models. This was carried out by means of the digital cell image analysis of Feulgen-stained nuclei. Two to three hundred cell nuclei were analyzed for each of the 324 samples under study. The three cell lines were submitted to experimental conditions including the addition of dihydrotestosterone (DHT), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF), either alone or in combination, to the culture media. The results show that under the present culture conditions, the PC3 cell line was DHT-, EGF- and bFGF-insensitive. In contrast to what is generally reported in the literature, the DU145 cell line was DHT- and EGF-sensitive under the present culture conditions, but bFGF-insensitive. The LNCaP cell line was DHT-sensitive, but EGF- and bFGF-insensitive. While mainly tetraploid, the three cell lines nevertheless exhibited a significant level of heterogeneity in their nuclear DNA content distributions. Indeed, the proportions of non-tetraploid (diploid, hyperdiploid, triploid, hypertriploid, hypertetraploid, polymorphic) DNA histograms were 14% in the PC3, 16% in the DU145, and 29% in the LNCaP cell lines. These results suggest that the DNA ploidy level would not influence the hormone sensitivity level in the cell lines since they had significantly distinct hormone sensitivity profiles while remaining mainly tetraploid.

Mxt mammary-tumor treatment with a high-temperature radiofrequency ablation device.

A special catheter with a deploying needle was used to treat MXT mouse mammary tumors transplanted onto the flanks of B6D2F1 mice. The catheter was connected to a radiofrequency generator. Treatment was applied for five minutes, with central tumor temperatures reaching over 100 degrees C. Histopathological examination revealed extensive localized, reproducible and controlled necrosis of the tumors in animals sacrificed at days 5 and 15 after treatment. The effectiveness of this device against MXT tumor growth was enhanced when a combination of radiofrequency and intraperitoneal chemotherapy (9 injections of adriamycin, etoposide and cyclophosphamide) was administered. This study demonstrates the effectiveness and reproducibility of tumor destruction and the increase in effectiveness of chemotherapy effects by radiofrequency heating.

Influence of culture media on the morphological differentiation of the PC-3 and DU145 prostatic neoplastic cell lines.

The concept of differentiation of prostate cancer in terms of morphonuclear characteristics and population dynamics was investigated on the PC-3 and DU145 cell lines. A software based on the concept of Voronoi paving was set up in order to characterize the structure of these cell lines growing in vitro on histological slides. The morphonuclear characteristics were assessed by means of the digital cell image analyses of Feulgen-stained nuclei. The in vitro "morphonuclear" and "pseudo-tissular" differentiations of the PC-3 and DU145 cells were described in terms of the use of various culture media, i.e., media supplemented with either 10% (F10 medium) or 1% (F1 medium) fetal calf serum and with (or without) platelet-derived growth factor and dihydrotestosterone (PA10 and PA1 media). The present data reveal that the PC-3 cell line would be more hormone-sensitive than the DU145 one. Indeed, decreasing the FCS concentration in the culture medium while adding DHT and PDGF led to marked modifications to the morphonuclear characteristics of the PC-3 cells, but not to the DU145 cells. These modifications corresponded to an increase in nuclear size occurring concomitantly with chromatin decondensation. In the same way, spectacular modifications in terms of medium-induced pseudo-tissular differentiation were observed in the PC-3 cell line, but not in the DU145 one. Such modifications corresponded to an increase in clone size related to an increase in the mean distances between neighboring cell nuclei in a given clone. Thus, according to the criteria defined in this study, the PC-3 cell line would seem to maintain a higher degree of differentiation than the DU145 cell line.

Suppression of preovulatory luteinizing hormone surges in heifers after intrauterine infusions of Escherichia coli endotoxin.

A study was conducted to test the hypothesis that high cortisol concentrations associated with products of infections (endotoxin) cause derangement in the neuroendocrine mechanism controlling ovulation in heifers. Eight Holstein heifers were given 2 injections of prostaglandin (PG), 11 days apart, to synchronize estrus. Starting from 25 hours after the second injection of PG (PG-2), the uterus of each heifer was infused with 5 ml of pyrogen-free water (control, n = 3) or Escherichia coli endotoxin (5 micrograms/kg of body weight) in 5 ml of pyrogen-free water (treated, n = 5), once every 6 hours for 10 treatments. Blood samples were obtained every 15 minutes via indwelling jugular catheter for an hour before and 2 hours after each infusion, then hourly until an hour before the next infusion. Ultrasonography of the ovaries was performed every 12 hours, starting 24 hours after PG-2 injection until 96 hours after PG-2 injection. Serum concentrations of luteinizing hormone and cortisol were determined by validated radioimmunoassays. Changes in cortisol concentrations were not detected in control heifers with preovulatory luteinizing hormone surges at 60 to 66 hours after PG-2 injection, followed by ovulations 72 to 96 hours after PG-2 was injected. None of the treated heifers ovulated, and the resulting follicular cysts (14 to 18 mm diameter) persisted for 7 to 21 days. In all treated heifers, serum cortisol concentrations increased (4- to 10-fold) during the first 2 hours after each infusion and then decreased gradually until the next infusion. Luteinizing hormone concentrations remained at baseline values throughout the treatment period in all treated heifers.(ABSTRACT TRUNCATED AT 250 WORDS)

Short-term changes in serum luteinizing hormone, ovarian response and reproductive performance following gonadotrophin releasing hormone treatment in postpartum dairy cows with retained placenta.

Thirty-six postpartum Holstein cows consisting of eighteen cows that shed the placenta soon after calving (NRP) and eighteen cows that retained placenta greater than 24 h (RP) were used. There were four treatment groups. Group I consisted of 9 NRP cows which received intramuscular injection of sterile saline on day 15 postpartum. The second group consisted of 9 NRPN cows which received 100 micrograms of gonadotrophin releasing hormone on day 15 postpartum (NRPT). The third group consisted of 9 RP cows which received saline on day 15 postpartum (RPN) and the fourth group consisted of 9 RP cows which received 100 micrograms of gonadotrophin releasing hormone on day 15 postpartum (RPT). Blood samples were collected once daily during the first month and once every other day during the second month postpartum. In addition fourteen cows (RPT, n = 7; NRPT, n = 7), were used to study short-term changes in serum luteinizing hormone concentrations following gonadotrophin releasing hormone treatment on day 15 postpartum. Blood samples were obtained from these cows every 15 min during 1 h before and 6 h after gonadotrophin releasing hormone administration. Sera from all samples were assayed for progesterone and luteinizing hormone concentrations. Starting from four days after calving rectal palpations and ultrasound examinations of the ovaries were carried out once every four days until day 28 postpartum in order to monitor ovarian changes. All cows were inseminated on the first estrus after day 60 postpartum and examined for pregnancy between 35 and 42 days after insemination.(ABSTRACT TRUNCATED AT 250 WORDS)