Glucosylceramide synthase protects glioblastoma cells against autophagic and apoptotic death induced by temozolomide and Paclitaxel.

Glioblastoma is a deadly cancer with intrinsic chemoresistance. Understanding this property will aid in therapy. Glucosylceramide synthase (GCS) is associated with resistance and poor outcome; little is known about glioblastomas. In glioblastoma cells, temozolomide and paclitaxel induce ceramide increase, which in turn promotes cytotoxicity. In drug-resistant cells, both drugs are unable to accumulate ceramide, increased expression and activity of GCS is present, and its inhibitors hinder resistance. Resistant cells exhibit cross-resistance, despite differing in marker expression, and cytotoxic mechanism. These findings suggest that GCS protects glioblastoma cells against autophagic and apoptotic death, and contributes to cell survival under chemotherapy.

Liver tumors induced by a new beta-adrenoreceptor blocking agent in female rats.

A newly synthesized beta-adrenoreceptor blocking agent, DL-1-(2-nitro-3-methyl-phenoxy)-3-tert-butylamino-propan-2-ol (ZAMI 1305), administered intragastrically for 6 months induced hepatocellular carcinomas in female outbred Wistar rats in a dose-dependent fashion but was harmless to the livers of male outbred Wistar rats. Experiments with castrated animals confirmed the dependence of the carcinogenic effect of ZAMI 1305 on the hormonal status of the rat. When an isomer of ZAMI 1305, DL-1-(2-nitro-5-methyl-phenoxy)-3-tert-butylamino-propan-2-ol (ZAMI 1327), and DL-practolol, which have pharmacologic activities similar to those of ZAMI 1305, were administered by the same experimental technique as used for ZAMI 1305, no liver tumors appeared either in male or female rats.

Formation of bioactive sphingoid molecules from exogenous sphingomyelin in primary cultures of neurons and astrocytes.

Exogenous sphingomyelin, radiolabelled at the sphingosine moiety, was administered to primary cultures of cerebellar granule cells and astrocytes for different pulse times (20 min-2 h) and the fate of the radioactivity was followed. Ceramide was the main metabolic product in both cells, whereas sphingosine, glucosyl-ceramide and gangliosides GM3 and GD3 were produced only in astrocytes. When endocytosis was prevented and the lysosomal apparatus inactivated, ceramide formation was reduced slightly in granule cells and almost completely blocked in astrocytes, with disappearance of sphingosine, glucosyl-ceramide, GM3 and GD3. These data indicate that (a) ceramide is rapidly produced in cerebellar granule cells and astrocytes, presumably at the level of the plasma membrane in the first cell type, and of the lysosomes in the second one; (b) sphingosine is produced in cerebellar astrocytes by lysosomal sphingomyelin degradation and is partly reused for glucosyl-ceramide and ganglioside biosynthesis.

Formation of free sphingosine and ceramide from exogenous ganglioside GM1 by cerebellar granule cells in culture.

Cerebellar granule cells differentiated in culture were incubated with ganglioside [3H-Sph]GM1 in order to have it inserted into the plasma membrane and metabolized. Among the formed metabolites radioactive sphingosine and ceramide were identified. [3H]Ceramide started to be measurable after 10 min of incubation (pulse), and [3H]sphingosine after 15 min. Their concentrations increased with pulse time, and, after a 1-hour pulse, with chase time. After a 1-hour pulse with 2 x 10(-6) M [3H-Sph]GM1 followed by a 4-hour chase, the amount of [3H]sphingosine and [3H]ceramide formed were 0.04 and 0.4 pmol/10(6) cells, respectively. Particularly the ability to produce sphingosine was higher in differentiated than in undifferentiated cells. It is concluded that ganglioside turnover contributes to the maintenance of the intracellular levels of free sphingosine and ceramide.

Cerebellar granule cells in culture exhibit a ganglioside-sialidase presumably linked to the plasma membrane.

Cerebellar granule cells differentiated in culture were incubated with ganglioside [3H-Sph]GD1a in order to have it inserted into the plasma membrane, internalized by endocytosis, and metabolized. The metabolites formed included GM1, product of GD1a desialosylation. No GM1 or other metabolites were present in the incubation medium, whereas with the lysosomal apparatus blocked by chloroquine, or GD1a endocytosis prevented at 4 degrees C, the only metabolite formed was GM1. These results suggest that GD1a desialosylation did not occur either extracellularly or intracellularly but likely, at the membrane level. Similar results were obtained with [3H-Gal]GD1b, whereas no degradation of [3H-NeuAc]GM1 took place in the presence of chloroquine or at 4 degrees C. In conclusion, cerebellar granule cells express in vivo a sialidase, presumably located on the cell surface, that affects GD1a and GD1b but not GM1.

Involvement of a ceramide activated protein phosphatase in the differentiation of neuroblastoma Neuro2a cells.

The possible involvement of protein phosphatase in ceramide-mediated neural cell differentiation was investigated. Neuroblastoma Neuro2a cell differentiation induced by retinoic acid, or conditions causing an increase in cellular ceramide, was significantly inhibited by the serine/threonine phosphatase inhibitor okadaic acid, at concentrations as low as 2.5 nM. A crude cytosolic preparation from Neuro2a cells was found to have a cation-independent protein phosphatase activity that was stimulated by ceramide in a dose-dependent manner. Short- and long-chain ceramides, but not sphingosine and related dihydro-derivatives, were active. Ceramide-activated protein phosphatase activity from Neuro2a cells was inhibited by 5 nM okadaic acid. The data indicate that a type 2A protein phosphatase is involved in ceramide-mediated differentiation of Neuro2a cells.

Salvage of catabolic products in ganglioside metabolism: a study on rat cerebellar granule cells in culture.

Cerebellar granule cells in culture were subjected to a pulse (0.5-4 h)-chase (0-4 h) of 10(-6) M [3H]ganglioside GM1 carrying the radioactive label at the level of NeuAc ([3H-NeuAc]GM1), Sph ([3H-Sph]GM1) or Gal ([3H-Gal]GM1) and the formed [3H]metabolites were determined. With all forms of [3H]GM1, there was formation of [3H]catabolites, including [3H]H2O and [3H]biosynthetic products obtained by recycling of [3H]NeuAc, [3H]Sph and [3H]Gal released during intralysosomal ganglioside degradation (salvage processes). Much higher amounts of [3H]H2O were produced from [3H-Gal]GM1 than [3H-Sph]GM1 and [3H-NeuAc]GM1; conversely, more products from salvage processes (polysialogangliosides GD1a, GD1b, GT1b, O-acetylated GT1b, protein-bound radioactivity) were obtained with [3H-NeuAc]GM1 than the two other forms of [3H]GM1. Liberated [3H]NeuAc produced 10-fold less tritiated water and 10-fold higher salvage products than [3H]Gal. Using [3H-NeuAc]GM1, granule cells appeared to metabolize 7.7% of membrane-incorporated exogenous GM1 per hour with a high degree of NeuAc recycling and the calculated metabolic half-life was 6.5 h.

Metabolism of exogenous ganglioside GM1 in cultured cerebellar granule cells. The fatty acid and sphingosine moieties formed during degradation are re-used for lipid biosynthesis.

Cerebellar granule cells, differentiated in vitro, were parallelly fed with [Sph-3H]GM1 and [stearoyl-14C]GM1, under identical conditions (10(-6) M ganglioside; pulse, from 1-4 h; chase, up to 24 h after 4 h pulse) and the salvage pathways of sphingosine and stearic acid were investigated. It was observed that both sphingosine and stearic acid, liberated during the intralysosomal degradation of ganglioside, are metabolically recycled, along distinct pathways. Sphingosine is used for the biosynthesis of a number of sphingolipids, particularly ceramide, glucosyl-ceramide, gangliosides and sphingomyelin; stearic acid is utilized for the biosynthesis of sphingolipids, and to a greater extent, glycero-phospholipids, especially those endogenously richer in stearic acid (phosphatidyl-ethanolamine and phosphatidyl-choline). No evidence was provided for a salvage pathway for ceramide.

GM3 ganglioside inhibits endothelin-1-mediated signal transduction in C6 glioma cells.

We found that sparse and confluent C6 glioma cells differ both in GM3 content, which increases with cell density, and in endothelin-1 (ET-1)-induced phosphoinositide hydrolysis, which was markedly higher in the sparse cells than in the confluent. Also after manipulation of the cellular GM3 content through treatment with exogenous GM3 or with drugs known to affect GM3 metabolism, the ET-1 effect was inversely related to GM3 cellular levels. Cell treatment with an anti-GM3 mAb resulted in the enhancement of ET-1-induced phospholipase C activation and restored the capacity of GM3-treated cells to respond to ET-1. These findings suggest that the GM3 ganglioside represents a physiological modulator of ET-1 signaling in glial cells.

Behaviour of nitric oxide synthase in rat cerebellar granule cells differentiating in culture.

The possible relation between nitric oxide synthase (NOS) activity and neural differentiation was investigated using primary cultures of rat cerebellar granule cells differentiating in culture. NOS activity was measured in the cytosolic and particulate fractions obtained from cell homogenate. In the experimental conditions used the optimal pH for NOS activity was about 6.4, the activity being about 3-fold higher than at pH 7.4. Cerebellar granule cell differentiation was associated with marked increases in NOS activity. In undifferentiated cells the enzyme was almost evenly distributed between the cytosolic and particulate fractions, during differentiation there was a 12-fold increase in activity in the cytosolic enzyme and a 3-fold increase in the particulate one. This indicates a marked preferential enrichment of the cytosolic enzyme during differentiation. Cerebellar granule cells produced and released NO in the culture medium; NO formation being markedly higher in differentiated cells (7-12 DIC) than in undifferentiated (2-3 DIC) ones. These data demonstrate a relationship between NOS expression and NO production and the differentiation of cerebellar granule cells, supporting the notion that NO may play a role in this process.

Sphingosine inhibits nitric oxide synthase from cerebellar granule cells differentiated in vitro.

The effects of different bioactive sphingoid molecules on NOS activity of differentiated cerebellar granule cells were investigated by measuring the conversion of [3H]arginine to [3H]citrulline. Cytosolic Ca2+-dependent NOS activity was strongly inhibited in a dose-dependent manner by sphingosine in concentrations of 1-40 microM. This inhibition seems to be peculiar to sphingosine in that ceramide, N-acetylsphingosine, sphingosine-1P, sphinganine and tetradecylamine have no effect on the cytosolic enzyme at the considered concentrations, suggesting that it is the bulk of the sphingosine hydrophilic portion that is critical for cytosolic NOS inhibition. This inhibition of cytosolic NOS is not reversed by increasing the arginine concentration, so a competitive mechanism can be excluded. Instead, increasing the concentrations of calmodulin led to loss of sphingosine inhibition, suggesting that sphingosine interferes with the calmodulin-dependent activation of the enzyme by a competitive mechanism. Sphingosine and related compounds had no effect on the particulate Ca2+-independent NOS activity. The data obtained suggest that sphingosine could be involved in the regulation of NO production in neurons.

Nitric oxide production in living neurons is modulated by sphingosine: a fluorescence microscopy study.

An investigation was carried out into the possible effect of sphingosine (Sph) on nitric oxide (NO) production in living neurons. Differentiated granule cells were used in a dynamic videoimaging analysis of single cells labeled, simultaneously, with FURA-2 and the NO indicator 4,5-diaminofluorescein. The results demonstrate that Sph exerts a potent inhibitory effect on the Ca2+-dependent production of NO, without modifying the [Ca2+]i. The effect appears to be specific as neither ceramide nor Sph-1-phosphate had any effect on the NO and [Ca2+]i levels. The data demonstrate that Ca2+-dependent NO production is a specific Sph target in living granule cells, suggesting that this bioactive sphingoid plays a relevant role in neuronal NO signaling.

Changes in the activity and distribution of the ribosomal dissociation factor of rat liver during growth.

In order to assess the relationships between the rate of growth and the activity of the initiation factors of protein synthesis, the activity and intracellular distribution of partially purified preparations of the ribosomal dissociation factor, obtained from the 0.5 M KCl wash of the ribosomes and from the high-speed supernatant (cytosol) of rat liver, were investigated in animals aged 1, 30, 60, 90 and 180 days. During the early phases of growth the activity of the dissociation factor was found mostly in the KCl wash of ribosomes. With advancing age, this activity decreased whereas that of the supernatant was found to increase. The activity of the supernatant was similar to that of ribosomes at 60, 90 and 180 days of age. The older rats showed, however, a decline in the activity of the dissociation factor. These observations suggest an increased rate of protein synthesis initiation in the early phases of growth in rat liver. With advancing age, a progressive reduction of ribosome recycling into subunits might take place, due to the increased accumulation of the dissociation factor in the cytosol. This might suggest a lowering of the initiation reactions of protein synthesis in such conditions.

Salvage pathways in glycosphingolipid metabolism.

In this review, the focus is on the role of salvage pathways in glycosphingolipid, particularly, ganglioside metabolism. Ganglioside de novo biosynthesis, that begins with the formation of ceramide and continues with the sequential glycosylation steps producing the oligosaccharide moieties, is briefly outlined in its enzymological and cell-topological aspects. Neo-synthesized gangliosides are delivered to the plasma membrane, where their oligosaccharide chains protrude toward the cell exterior. The metabolic fate of gangliosides after internalization via endocytosis is then described, illustrating: (a) the direct recycling of gangliosides to the plasma membrane through vesicles gemmated from sorting endosomes; (b) the sorting through endosomal vesicles to the Golgi apparatus where additional glycosylations may take place; and (c) the channelling to the endosomal/lysosomal system, where complete degradation occurs with formation of the individual sugar (glucose, galactose, hexosamine, sialic acid) and lipid (ceramide, sphingosine, fatty acid) components of gangliosides. The in vivo and in vitro evidence concerning the metabolic recycling of these components is examined in detail. The notion arises that these salvage pathways, leading to the formation of gangliosides and other glycosphingolipids, sphingomyelin, glycoproteins and glycosaminoglycans, represent an important saving of energy in the cell economy and constitute a relevant event in overall ganglioside (or glycosphingolipid, in general) turnover, covering from 50% to 90% of it, depending on the cell line and stage of cell life. Sialic acid is the moiety most actively recycled for metabolic purposes, followed by sphingosine, hexosamine, galactose and fatty acid. Finally, the importance of salvage processes in controlling the active concentrations of ceramide and sphingosine, known to carry peculiar bioregulatory/signalling properties, is discussed.

Polyamine concentration, activity of the ribosomal dissociation factor and proportion of ribosomes active in protein synthesis in the 4-dimethylaminoazobenzene-induced rat hepatoma.

4-Dimethylaminoazobenzene (DAB) induced liver tumour tissue showed a reduced proportion of ribosomes active in protein synthesis compared with control liver. Tumour cell extracts caused an increased association of ribosome subunits into inactive 80 S monomers, compared with the dissociation into active subunits caused by normal liver extracts. These findings may be explained, at least in part, by the increased proportion of spermidine to putrescine found in tumour tissue which would predispose towards association of the subunits.

Effects of peripheral sympathetic denervation induced by guanethidine administration on the mechanisms regulating puberty in the female guinea pig.

The effects of peripheral sympathetic denervation induced by guanethidine administration to newborn and 10-day-old female guinea pigs on puberty, ovulation and the follicular population were analysed. Peripheral sympathetic denervation beginning at birth resulted in the loss of ovarian norepinephrine content (0.95. +/- 0.1 ng/mg wet tissue in untreated control animals vs non detectable in treated animals). Guanethidine administration to newborn or 10-day-old guinea pigs advanced puberty (age of vaginal opening: 27 +/- 1.2 days (newborn), 26 +/- 1.7 (10-day-old) vs 37 +/- 0.7 (control), P < 0.001) and ovulation. The number of corpora lutea in control and denervated animals was similar (3.5 +/- 0.2 vs 3.3 +/- 0.3). The relative weight (mg/100 g body weight) of the ovaries and adrenals in the denervated animals autopsied during the late follicular phase (24-48 h after vaginal opening) increased (ovaries: 27.8 +/- 1.3, 28.9 +/- 3.0 vs 20.9 +/- 0.8, P < 0.05; adrenals 36.4 +/- 1.4, 37.0 +/- 0.8 vs 31.6 +/- 1.5, P < 0.05), while the uterine weight diminished (179 +/- 13, 149 +/- 28 vs 292 +/- 20). When the animals were killed during the late luteal phase (9-11 days after vaginal closure), the relative weight of the ovaries of newborn guanethidine-treated animals was higher than that of the control animals (21.4 +/- 1.7 vs 16.8 +/- 1.4, P < 0.05). The mean number of follicles counted in the ovaries of denervated animals was significantly higher than in control animals (1736 +/- 230 vs 969 +/- 147, P < 0.05). The mean diameter of the follicles in the untouched control ovary in animals killed in the late follicular phase was significantly larger than from animals killed in the late luteal phase (263 +/- 3.9 microns vs 248 +/- 3.0 microns, P < 0.01). The mean diameter of the follicles measured in the ovaries of denervated animals was significantly higher than in controls (animals treated from birth 274 +/- 2.0 microns vs 255 +/- 2.4, P < 0.05; animals treated from day 10, 286 +/- 2.3 microns vs 257 +/- 2.3, P < 0.05). When the mean diameter of the follicles in the left and right ovary of the untouched control was analysed, the follicular diameter in the left ovary was significantly larger than in the right ovary (309 +/- 6.0 microns vs 214 +/- 3.9, P < 0.01); the response of the left and right ovaries to sympathetic denervation was the opposite. The results obtained in the present study suggest that ovarian innervation plays a role in the regulation of follicular growth, maturation and atresia which is not related to changes in steroid secretion by the ovary, but to other regulatory mechanisms.

Is there a cholinergic circadian rhythm throughout the oestrous cycle related to ovulation in the rat?

Rats with a 4-day oestrous cycle were given a single dose of atropine (100, 300, 500 or 700 mg/kg body wt) at 13.00 h on the days of oestrus, dioestrus 1, dioestrus 2 or pro-oestrus and were autopsied on the next expected day of oestrus. The doses of atropine (in mg/kg body wt) necessary to block ovulation during the cycle were 300 at oestrus, 100 at dioestrus 1 or 2 and 700 at pro-oestrus. A single dose of atropine (100 mg/kg) at oestrus, dioestrus 1 or dioestrus 2 was given at 09.00, 13.00, 17.00 or 21.00 h, autopsy again being performed on the next expected day of oestrus. The ability of atropine to block ovulation appeared to have a circadian rhythm, with a maximum blockade at 13.00 h on dioestrus 1 and dioestrus 2 and a minimum at 21.00 h on the same days. Hormone replacement (human chorionic gonadotrophin at oestrus, dioestrus 1 or 2, oestradiol benzoate at dioestrus 2 or progesterone at pro-oestrus) re-established normal ovulation in rats whose ovulation was blocked with atropine (100 mg/kg) on dioestrus 1 at 13.00 h. When ovulation was blocked with atropine but no hormone replacement had been given, rats ovulated 24 h after the next expected day of oestrus. Results obtained in these experiments suggest the existence of a circadian rhythm of gonadotrophin secretion throughout the oestrous cycle and a close relationship between that rhythm and the cholinergic system.

Effects of unilateral destruction of the cervico-vaginal plexus on ovulation in the rat.

Hemidestruction of the cervico-vaginal plexus in 4- or 5-day cyclic rats were performed. After recovery, all animals had normal oestrous cycles. At oestrus itself, however, the number of ova released by lesioned animals was respectively 23% and 50% less than in the sham-operated and intact control rats. The possible participation of peripheral innervation in normal ovarian function is discussed.

Hysterectomy of the newborn guinea-pig, subsequent effects on the oestrous cycle and life span of the corpora lutea.

Longer oestrous cycles result from neonatal hysterectomy than from hysterectomy in adult life. Section and cauterization of the utero-vaginal union also prolonged the vaginal closure period up to an average of 55 days. The destruction of the mesometrium did not lengthen the oestrous cycle. Uterine autografts in hysterectomized newoborn guinea-pigs did not prevent the long cycles.

A non-steroidal gametic factor linked to DNA modulates delta-aminolevulinic acid synthase inducibility acting on liver transcriptional and translational processes.

The effect of an acidic factor of low molecular weight (about 1,000 daltons), extracted from bovine spermatozoan DNA, on the inducibility of delta-aminolevulinic acid synthase by ethanol during aging in rat has been examined. The increased enzyme inducibility in 600-day old rats is supported by stimulation of transcriptional and translational processes; on the contrary, in 30-day old rats, the higher enzyme values induced by ethanol are significantly decreased after factor treatment. The active factor is strongly DNA-bound in the native spermatozoan DNA. This would imply a possible role in regulating gene expression in vivo.