Pierre N.M. Konings

Reversal by NGF of cytostatic drug-induced reduction of neurite outgrowth in rat dorsal root ganglia in vitro.

Cytostatic drugs, like cisplatin, vincristine and taxol, when given to cancer patients may cause peripheral neuropathies. We were interested in the potential neuroprotective effects of neurotrophic factors against such neuropathies. To this aim we studied the effects of these cytostatic agents on sensory fibers located in the dorsal root ganglia (DRG) in vitro and studied whether nerve growth factor (NGF) could reverse the cytostatic induced morphological changes on intact DRG (1 DRG/well, n = 10 per dose). Neuritogenesis from DRG was measured with an image analysis system following exposure to different concentrations of cytostatic drugs in the presence of 3 ng NGF/ml and cytosine arabinoside (Ara-C, 10(-6) M). Relative neurite outgrowth in intact DRG in culture was reduced dose-dependently, (a) by vincristine starting at a dose of 0.4 ng/ml for 2 days (-33% as compared to control; P < 0.001, Students t-test); (b) by taxol 10 ng/ml (-60%; P < 0.001), and (c) by cisplatin 3 micrograms/ml (-47%, P < 0.001). Cisplatin also prevented the migration of satellite cells away from the intact DRG along the extending neurites into the well in contrast to control, vincristine, or taxol. To evaluate the neuroprotective potential of NGF in this in vitro cytostatic neuropathy model, we incubated intact DRG with cytostatic agents in combination with increasing amounts of NGF. Neurite outgrowth from DRG treated with vincristine (0.5 ng/ml)+NGF (3 ng/ml) for 2 days was significantly higher (+87%) than after treatment with vincristine + 1 ng NGF/ml (P < 0.001). Neurite outgrowth from DRG treated with taxol (20 ng/ml)+NGF (3 ng/ml) for 2 days was significantly higher (+228%) than after taxol + 1 ng NGF/ml (P < 0.05). Neuritogenesis from DRG treated with cisplatin (2.5 micrograms/ml)+NGF (3 ng/ml) for 2 days was significantly increased (+105%) compared to treatment with cisplatin + 1 ng NGF/ml (P < 0.001). DRG thus appear to be a very suitable model for studying cytostatic drug-induced neuropathies in vitro and NGF has a clear neuroprotective effect on the vincristine-, taxol-, and cisplatin-induced neuropathies in this in vitro model.

SR 57746A attenuates cytostatic drug-induced reduction of neurite outgrowth in co-cultures of rat dorsal root ganglia and Schwann cells

A co-culture system of intact rat dorsal root ganglia (DRG) with Schwann cells was used to evaluate the potential neurotrophic activity of SR 57746A. Neuritogenesis from DRG was measured with an image analysis system following exposure to different concentrations of SR 57746A. Neurite outgrowth of intact DRG was increased by SR 57746A and this was more obvious in the presence of co-cultured Schwann cells. The neuroprotective properties of SR 57746A were studied in co-cultures of DRG and Schwann cells, in which neuritogenesis was reduced by the cytostatic drugs cisplatin, vincristine and taxol. It was found that neurite outgrowth from DRG treated with cisplatin (3 micrograms/ml) and 10 microM SR 57746A for 3 days was significantly higher than after treatment with cisplatin alone. Similarly, neuritogenesis from DRG treated with taxol (0.01 microgram/ml) or vincristine (0.5 ng/ml) in combination with 10 microM SR 57746A was significantly increased compared to treatment with taxol or vincristine alone. When intact DRG were incubated in vitro with 3 micrograms/ml cisplatin and without Schwann cells, 10 microM SR 57746A also had a neuroprotective effect. These data suggest that SR 57746A has neuroprotective potential and that this effect does not depend solely on the presence of Schwann cells.

Synthesis of sialic acid-lipid conjugates and their neuritogenic effects on N1E.115 neuroblastoma cells☆

Several sialic acid-lipid conjugates having one or more sialic acid residues were prepared via phosphite and methylthiomethyl intermediates. The synthetic compounds were found to promote neurite outgrowth on N1E.115 neuroblastoma cells. In particular, the trisialocholesterol derivative 19 exhibited potent neuritogenic activity.

α-Sialyl cholesterol increases laminin in Schwann cell cultures and attenuates cytostatic drug-induced reduction of laminin

Schwann cells play an important role in peripheral nerve regeneration. Here, we report the effect of alpha-sialyl cholesterol (alpha-SC), a derivative of the sialic acid-containing natural gangliosides, and the cytostatic agents, cisplatin, taxol and vincristine on the laminin production in Schwann cell cultures isolated from rat sciatic nerves. Laminin, one of several extracellular matrix components produced by Schwann cells, is known to potentiate axonal outgrowth. Laminin content was increased by alpha-SC, starting at 7.0 micrograms/ml with a maximal effect at 22.4 micrograms/ml (30%, P < 0.001). The three cytostatic drugs, dose-dependently reduced laminin content in Schwann cell cultures: (1) cisplatin at a threshold dose of 2 micrograms/ml (-26.4%, P < 0.001); (2) taxol, starting at a dose of 1 ng/ml (-8.0%, P < 0.05); and (3) vincristine, starting at 0.5 ng/ml (-5.9%, P < 0.05). Cultured Schwann cells were incubated with cytostatic drugs in combination with increasing amounts of alpha-SC and it was found that, depending on the cytostatic drug concentration used, alpha-SC could reduce or completely prevent the cytostatic drug-induced reduction of laminin in Schwann cell cultures. Co-treatment with alpha-SC also reduced part of the morphological changes caused by the cytostatic drugs. alpha-SC did not counteract the anti-proliferative effect of the cytostatic drugs on K-562 human erythroleukemia cells. In conclusion, alpha-SC increased laminin content in Schwann cell cultures and protected Schwann cell cultures against the decrease of laminin by cytostatic drugs without interfering with the anti-proliferative potential, suggesting that alpha-SC may have clinical use in protecting cancer patients against the neurotoxic effects of cytostatic drugs.

Combined in situ hybridisation, northern blot analysis, and receptor binding studies in clones expressing different levels of the human 5-HT1A receptor

In order to set up the technique of semi-quantitative in situ hybridisation to detect the serotonin receptor mRNA levels in brain tissue, a panel of three Swiss 3T3 cell clones (named clones 66, 53 and 47) expressing the human 5-HT1A receptor at different densities were used as a model. The clones were generated by limiting dilution from pools of stably transfected cells. In addition membranes were prepared from each clone to perform receptor binding studies. Clones 66, 53, and 47 showed saturable binding for the agonist [3H]-8-OH-DPAT, with receptor densities (Bmax) of 227 +/- 86, 548 +/- 107 and 1505 +/- 212 fmol/mg protein respectively, and with corresponding affinity constants (pKd) of 8.8 +/- 0.1, 9.1 +/- 0.1, and 9.1 +/- 0.1 nM, respectively. Northern blot analysis using a specific probe for the 5-HT1A receptor revealed the presence of a single 1.56 kilobase mRNA species in the 5-HT1A receptor clones but not in control cells. In situ hybridisation studies were performed by measuring the 5-HT1A receptor mRNA levels in these three 5-HT1A transfectants using [35S]alphaCTP labeled riboprobes (sense and anti-sense). The following rank order of receptor mRNA expression was found for clones 66, 53 and 47 respectively: 0.140 +/- 0.001, 0.365 +/- 0.045 and 0.835 +/- 0.115 (relative optical density units). With the sense probe no specific labelling was observed. In conclusion, a positive correlation was found between receptor density (Bmax) and receptor mRNA expression (semi-quantitative in situ hybridisation) using human 5-HT1A receptor clones with different expression levels.

Morphometric analysis of cisplatin-induced neurite outgrowth in N1E-115 neuroblastoma cells

Cisplatin, a widely used cytostatic drug for the control of a variety of neoplastic tumors, unexpectedly induced neurite outgrowth in N1E-115 neuroblastoma cells and this phenomenon was studied further in detail with morphometric analysis. As expected, cisplatin dose-dependently reduced cell number. At the same time, however, cisplatin affected the morphology of the neuroblastoma cells that changed from small rounded cell bodies into large flat cell bodies with neurites. The neurite length/cell as a function of cisplatin concentration showed a bell-shaped curve. The maximal effect (1200% of control) on neurite length/cell was observed at 1 microgram/ml cisplatin. In conclusion, cisplatin induced cellular differentiation in N1E-115 neuroblastoma cells at and just above threshold doses for cytostatic activity.

Regulation of CCK mRNA in the human neuroepithelioma cell line SK-N-MCIXC in response to second messenger activators.

Regulation of cholecystokinin (CCK) expression was studied in the human neuroepithelioma cell line SK‐N‐MCIXC. The cells were treated with the phosphodiesterase inhibitor isobutyl‐methylxanthine and the tumor promoting phorbol ester, phorbol‐12‐myristate 13‐acetate; activators of the cyclic AMP (cAMP) and protein kinase C (PKC) second messenger pathways, respectively. Levels of CCK mRNA were determined after 6, 12 and 24 hour drug treatments, with Northern blot analysis using human CCK cDNA hybridization probes. Activation of both cAMP and PKC second messenger pathways increased CCK mRNA levels in SK‐N‐MCIXC cells. These results indicate that the levels of CCK mRNA in SK‐N‐MCIXC cells are regulated by cAMP and PKC dependent mechanisms.