Dean Sandquist

Morphological differentiation of a murine neuroblastoma clone in monolayer culture induced by dexamethasone.

Dexamethasone induces morphological differentiation in murine neuroblastoma cells in culture. The percentage of differentiated cells depends on the concentration of dexamethasone in the medium and duration of treatment. After drug removal (with or without replating), many cells maintain their differentiated phenotype. Dexamethasone-treated cells have larger soma and contain more protein. Dexamethasone also produces a concentration-dependent inhibition of population growth. Growth inhibition is complete by 2 days following treatment with dexamethasone 50 micrograms/ml. Complete growth inhibition is achieved prior to the complete expression of morphological differentiation. Androstenedione, testosterone, and 17-beta-estradiol--all steroids without glucocorticoid activity--have no differentiating effect, although they inhibit growth or cause cell death at higher concentrations.

Muscarinic cholinergic stimulation increases cyclic GMP levels in rat hippocampus.

Abstract— Muscarinic cholinergic agonists increase cyclic GMP levels in a number of neural tissues. Since the rat hippocampus receives a cholinergic innervation from the septum, we decided to test whether cyclic GMP levels of the rat hippocampus are increased by bethanechol, a muscarinic cholinergic agonist. Incubation of rat hippocampi with varying concentrations of bethanechol showed that the increase in cyclic GMP levels is concentration‐dependent, 500 pwbethanechol producing a maximum increase of 490% over control values. The bethanechol‐evoked increases were blocked by the muscarinic antagonist atropine, and were calcium‐dependent. It is concluded that at least some of the cells projecting to the rat hippocampus form muscarinic cholinergic synapses which act via a cyclic GMP‐dependent mechanism.

Dexamethasone induces increased catecholamine biosynthesis in cultured neuroblastoma.

Abstract The effect of dexamethasone on morphological and biochemical parameters of catecholamine biosynthesis in cultured murine neuroblastoma cells was determined. Treatment for 7 days with 25 μM dexamethasone produced a more intense and uniform catecholamine fluorescence as determined by fluorescence histochemistry using the glyoxylic acid technique. Treatment with dexamethasone also produced a two-fold increase in dopamine content and a threefold increase in tyrosine hydroxylase activity compared with controls. These results suggest that this potent glucocorticoid induces differentiation in cultured murine neuroblastoma, as measured by both biochemical and morphological techniques.

Glucocorticoids Increase Tyrosine Hydroxylase Activity in Cultured Murine Neuroblastoma

Abstract: Cyclic AMP and glucocorticoids appear to have a role in regulating the activity of tyrosine hydroxylase (TH), as well as the expression of “morphological differentiation” in murine neuroblastoma. Monolayer cultures of C‐1300 murine neuroblastoma (clone NBP2) were treated with the following compounds in ethanol: dexamethasone, triamcinolone acetonide, hydrocortisone, cortexolone, androstenedione, testosterone, estradiol‐17β; or with the phosphodiesterase inhibitor Ro20–1724 [4‐(3‐butoxy‐4‐methoxybenzyl)‐2‐imidazolidinone]. Treatment with either 200 μg/ml Ro20–1724 or 50 μg/ml dexamethasone produced significant increases in TH activity compared to alcohol controls (1.44 vs. 0.82 nmol 14CO2/mg protein/hr compared to 0.095). Triamcinolone acetonide or hydrocortisone also produced smaller, but significant, increases in TH activity compared to dexamethasone. When steroid activities were compared at 25 μm concentration and after 60 min of incubation (to maximize TH activity), triamcinolone acetonide was not as effective (62%) as dexamethasone. The relatively inactive glucocorticoid cortexolone produced a slight but significant increase, while the androgens androstenedione and testosterone and the estrogen estradiol‐17β were without effect.

Growth cones in differentiated neuroblastoma: A time-lapse cinematographic and electron microscopic study

SummaryGrowth cones of ‘differentiating’ neuroblastoma cells in monolayer culture were studied by time-lapse cinematography and electron microscopy. Morphological differentiation, and thus growth cone formation, was induced by the glucocorticoid dexamethasone. Growth cones lengthened gradually at an average rate of 30 μm/h, advancing in stages that involved alternating extensions and retractions of the filopodia and lamellar sheets. During neurite growth the cell body usually remained stationary. The ultrastructure of growth cones was typified by several filopodia, each containing a bundle of microfilaments, agranular endoplasmic reticulum, aggregates of large agranular vesicles lying adjacent to filopodia (previously termed vesicle-filled mounds), many dense-cored vesicles, 100–140 nm in diameter, microtubules, bizarre and distorted mitochondria, and scattered free ribosomes. Comparing the findings with previous ultrastructural accounts of growth cones of cultured ganglion cells, similarities outnumbered differences. The organization of the microfilament bundles and the abundance of free ribosomes were remarkable in the neuroblastoma cell as was the profusion of dense-cored vesicles which were most numerous in the proximal portion of the growth cone.

Age as a factor in 6-hydroxydopamine-induced plasticity in the hypothalamus

SummaryThe question of age as a possible factor influencing the regenerative response of catecholaminergic varicosities in the hypothalamus was investigated in the supraoptic commissure and the paraventricular, periventricular, and dorsomedial hypothalamic nuclei of rats that had received intraventricular injections of the neurotoxin 6-hydroxydopamine when they were (1) neonates, (2) young adults, or (3) senescent adults. After postneurotoxin survival for 4, 21, 56, or 180 days, the animals were perfused, and the hypothalamic tissue sections were cut and processed using a glyoxylic acid method for localizing catecholamines. Four days following neurotoxin administration, counts of fluorescent varicosities showed a significant loss of catecholamine varicosities in each of the four areas. Subsequently, at least partial restoration of numbers of catecholamine varicosities occurred in all hypothalamic areas in all three age groups. It is concluded that, following selective lesions induced by the neurotoxin 6-OH-DA, catecholamine varicosities were restored both in immature and mature groups. According to the evidence obtained experimentally, the rate of restoration was greater in the neonate group, whereas the percentage restoration attained varies according to the hypothalamic area studied and the age of the animal.

Acyltransferase activity and cytochemical localization in differentiating neuroblastoma

Acyltransferase (AT) enzyme activity was assayed biochemically and localized cytochemically in neuroblastoma monolayers to investigate the possible role of the enzyme in neuroblastoma differentiation and neurite extension. Treatment of cultures for 1 day with serum-free medium or Ro20-1724, a cyclic AMP phosphodiesterase inhibitor, induced neurite outgrowth but did not alter acyltransferase activity. Treatment for 4 days with dexamethasone or Ro20-1724 induced neurite outgrowth and a doubling of enzyme activity per cell. Chromatographic separation of lipid classes indicated that dexamethasone enhanced triacylglyceride synthesis. Acyltransferase was localized in mitochondria of neuroblastoma cells. The results show that 1) dexamethasone stimulates the storage lipid metabolic pathway in neuroblastoma cells and 2) increased acyltransferase activity is concomitant with dexamethasone-induced morphological differentiation. However, AT activity and neurite extension may not be causally related.

Reduced tumorigenicity of cultured neuroblastoma cells after treatment in Vitro with dexamethasone

The effect of dexamethasone on tumorigenicity of cultured neuroblastoma and on de novo synthesis of DNA and protein was determined. Within 12 hr dexamethasone caused a dose-dependent inhibition of [3H]-thymidine incorporation into DNA. Incorporation of [3H]-leucine into protein was not affected by dexamethasone. Neurite formation was interrupted by actinomycin D or cycloheximide. Cells treated with dexamethasone before inoculation into A/J mice produced fewer tumors with longer latent periods than controls. About 2.6 times as many neuroblastoma cells treated with 50 micrograms/ml dexamethasone for 4 days were required for tumor development in 50% of recipient animals as compared to controls. Reduced tumorigenicity was dependent upon the length of treatment and the concentration of dexamethasone used. Cortexolone did not mimic the effects of dexamethasone. If, instead of inoculation, cells were replated and grown without dexamethasone, cellular aggregations appeared among the cells cultured in the absence of dexamethasone. By autoradiography, replated cells previously treated with ethanol displayed uniform incorporation of [3H]-thymidine, whereas replated cells from dexamethasone-treated cultures exhibited no incorporation in differentiated cells. However, incorporation was noted among the clusters. We hypothesize that tumors arising after dexamethasone treatment may be due to the presence of an unresponsive subpopulation of cells.

The guinea pig further demonstrates that rodent superior cervical ganglia differ from those of other species

Abstract Fluorescence microscopic examination of five hamster superior cervical ganglia demonstrated that there were 223 ± 31 small, intensely fluorescent (SIF) cells per ganglion, equivalent to 140 ± 26 SIF cells per milligram tissue (wet weight). Incubation of hamster superior cervical ganglia (SCG) in vitro with 50 μ m l -isoproterenol increased cyclic AMP concentrations to 622% of control values. Administration of 50 μ m dopamine was without effect. Comparison with other species suggests that the superior cervical ganglia of three rodents—the hamster, guinea pig, and rat—differ significantly from those of other species in their morphologic and biochemical properties.