Molecular mechanisms of GD3-induced apoptosis in U-1242 MG glioma cells.

An increasing amount of evidence indicates that the disialoganglioside GD3 is involved in apoptosis in many cell lines. Our previous studies demonstrated that endogenous GD3 expression induced apoptosis in U-1242 MG glioma cells transfected with the GD3 synthase gene (U1242MG-GD3 cells). In this paper, we present further investigations on the molecular mechanisms of GD3-induced apoptosis in this cell line. We found that endogenously synthesized GD3 localizes to the caveolae of this cell line, where it promotes the localization of death receptor 5 (DR5), tumor necrosis factor receptor-1 (TNF-R1), and Fas (Apo-1) to the caveolae. In addition, caspase-8 was translocated to the caveolar fraction and cleaved; the cleaved proteins were then re-located into the high density fractions. However, GD3 had no effect on the distribution of the adapter protein Fas-associated death domain (FADD). We conclude that GD3 functions as a regulatory molecule early in the extrinsic apoptosis pathway.

Diagnostic and prognostic value of glycosyltransferase mRNA in glioblastoma multiforme patients.

Glioblastoma multiforme (GBM) is the most common and aggressive primary human brain tumour in adults with an average survival of 11 months. The 2-year survival is less than 10%, and only a small proportion of patients are alive at 3 years. Despite improved treatment strategies and aggressive therapy, the prognosis of GBM has changed little in past decades. Thus, any test that can reliably and rapidly diagnose the tumour and predict patient survival could be a valuable tool. Herein we report the use of quantitative real-time polymerase chain reaction (PCR) to quantify five glycosyltransferase transcripts in gliomas. Our results indicate that measuring GM1 synthase (beta-1,3 galactosyltransferase) mRNA may provide a useful method for segregating GBMs from other types of gliomas. In these studies, 97% of gliomas (36/37 tumours) below a threshold value had a diagnosis of GBM compared with 49% (52/106 tumours) above the threshold. More importantly, the increased expression of GD3 synthase mRNA in combination with decreased GalNAcT message correlated with increased survival in 79 GBM patients (proportional hazards model controlling for age, P = 0.02). These data were further corroborated by a data analysis from one of our previous studies on gangliosides of 80 GBMs, in which increased amounts of GM3 and GD3 (which accumulate in the absence of GalNAcT) correlated with a longer survival (P < 0.01). Thus, measuring GalNAcT and GD3 transcripts may provide a rapid method to assess prognosis in GBM patients. In summary, the data indicate that measuring glycosyltransferase mRNA levels by real-time PCR may be clinically useful for determining both diagnosis and prognosis in GBM patients.

TRAIL-induced apoptosis in U-1242 MG glioma cells.

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induces apoptosis in U-1242 MG cells. To investigate the molecular events involved in this process, we studied the effects of TRAIL on the localization within membrane fractions of molecules critical to the extrinsic apoptotic pathway. We report here that death receptor-5 (DR5), tumor necrosis factor receptor-1 (TNF-R1), and Fas receptor (FasR) are all located in the caveolin-1-enriched membrane fractions, and TRAIL caused the translocation of DR5, FasR, and TNF-R1 to the caveolar fractions. Caspase-8 is mainly located outside of caveolae, but TRAIL caused it to redistribute to the caveolin-1-enriched fractions where it was cleaved. Within 6 hours, the cleaved caspase-8 appeared in the high-density, noncaveolin fractions. Using confocal microscopy, we found that DR5, caspase-8, and caveolin-1 became progressively concentrated in blebs of plasmalemma as they formed in response to TRAIL. Our results provide the first evidence for the caveolar localization of TNF-R1 and DR5 and the coordinated redistribution among membrane fractions of several death receptors in response to TRAIL. We propose that the coordinated movement of these molecules among membrane compartments is probably an important component of the mechanisms regulating and initiating the extrinsic apoptotic pathway in human glioma cells.

Endogenous GD3 ganglioside induces apoptosis in U-1242 MG glioma cells.

GD3 ganglioside induces apoptosis in several cell types, but the molecular events through which this occurs are largely unknown. We investigated the apoptotic effects of GD3 expression using U-1242 MG glioblastoma cells, as these cells synthesize almost exclusively GM3 and GM2 but not GD3. To express GD3 under the control of the TetOn system with minimum leakage, we modified the system by constructing a single tri-cistronic retrovirus vector containing three genes separated by two internal ribosome entry sites: (a) transcriptional silencer, tTS; (b) mutant of reverse transcriptional activator, rtTA2(S)-M2 (provided by H. Bujard, Heidelberg, Germany); and (c) enhanced green fluorescent protein (EGFP), as an indicator of the tri-cistronic gene expression. Using flow cytometry, we selected glioma cells (U1242MG-GD3 clone) that express high levels of GD3 in response to doxycycline. Expression of GD3 was associated with apoptosis as verified by annexin-V binding, TdT-mediated dUTPnick end-labelling assay (TUNEL), and EGFP degradation. GD3-induced apoptosis occurred via caspase-8 activation, as GD3 caused cleavage of caspase-8 and inhibition of caspase-8 activation by zlETD-fmk minimized GD3-induced apoptosis.

Mechanisms through which PDGF alters intracellular calcium levels in U-1242 MG human glioma cells.

PDGF-BB induces a rapid, sustained increase in intracellular calcium levels in U-1242 MG cells. We used several calcium channel blockers to identify the types of channels involved. L channel blockers (verapamil, nimodipine, nicardipine, nitrendipine and taicatoxin) had no effect on PDGF-BB induced alterations in intracellular calcium. Blockers of P, Q and N channels (omega-agatoxin-IVA, omega-conotoxin MVIIC and omega-conotoxin GVIA) also had no effect. This indicates that these channels play an insignificant role in supplying the Ca2+ necessary for PDGF stimulated events in U-1242 MG cells. However, a T channel blocker (NDGA) and the non-specific (NS) calcium channel blockers (FFA and SK&F 9365) abolished PDGF-induced increases in intracellular calcium. This indicates that PDGF causes calcium influx through both non-specific cationic channels and T channels. To study the participation of intracellular calcium stores in this process, we used thapsigargin, caffeine and ryanodine, all of which cause depletion of intracellular calcium stores. The PDGF effect was abolished using both thapsigargin and caffeine but not ryanodine. Collectively, these data indicate that in these human glioma cells PDGF-BB induces release of intracellular calcium from caffeine- and thapsigargin-sensitive calcium stores which in turn lead to further calcium influx through both NS and T channels.

Gangliosides inhibit PDGF-induced signal transduction events in U-1242 MG human glioma cells.

In this study we investigated the responses of intracellular calcium ([Ca2+]i) and protein kinase C (PKC) to PDGF in U-1242 MG cells. PDGF-BB stimulated [3H]PDBu binding approximately 2-3 fold. This response was inhibited by preincubating the cells with an inhibitor of phospholipase C (PLC), U73122, suggesting that PLC mediates the induction of PKC translocation by PDGF. PDGF also increased the concentration of [Ca2+]i that was attenuated in a calcium-free medium. This indicates that PDGF-induced elevation of [Ca2+]i is mainly due to influx of extracellular calcium. PDGF-stimulated translocation of PKC was inhibited by the intracellular calcium buffer BAPTA/AM. All gangliosides studied except GM3 inhibited these responses with similar efficacy. Collectively, these results indicate that the signal transduction pathway initiated by PDGF leading to PKC translocation in U-1242 MG cells is intact, and this pathway is inhibited by several gangliosides.

Gangliosides have a bimodal effect on DNA synthesis in U-1242 MG human glioma cells.

GM1, GD1a, and GT1b inhibit both PDGF-stimulated and serum-stimulated DNA synthesis in Swiss 3T3 cells and the human glioma cell line U-1242 MG in a dose-dependent manner. The ganglioside inhibitory effect is counteracted in a dose-responsive fashion by serum such that ganglioside-induced inhibition is essentially abolished in 10% serum. Because of the potentially important role that gangliosides play in growth regulation of human gliomas, this phenomenon was studied in detail using U-1242 MG cells. Stimulation of DNA synthesis by low doses of serum in U-1242 MG cells is inhibited in a dose-responsive fashion by ganglioside GM1. However, serum itself counteracts the inhibitory effect of ganglioside in a dose responsive way. Kinetic analyses demonstrate that GM1 competes with some components of serum for sites on U-1242 MG cells (Kb of GM1 = 12.5 microM). On the other hand, GM1, GD1a, and GT1b stimulate DNA synthesis in quiescent U-1242 MG cells in both sparse and confluent conditions, indicating that ganglioside-stimulated DNA synthesis is dependent on the phase of cellular growth rather than cellular density. This growth stimulatory effect of gangliosides is more potent on quiescent, confluent cells than quiescent, sparse cells. These results demonstrate that exogenously added gangliosides can have opposite (bimodal) effects on progression of human glioma cells through the cell cycle depending upon the growth phase of the cells.

Ganglioside modulation of the PDGF receptor. A model for ganglioside functions.

Gangliosides are a family of glycolipids that are present at the cell surface of all mammalian cells. Patterns of gangliosides are different in gliomas than normal brain, and exogenously added gangliosides affect the growth of cultured glioma cells. Gangliosides inhibit the activities of several kinases, including protein kinase C (PKC) and cAMP-kinase. U-1242 MG cells (derived from a human malignant glioma) have receptors for platelet-derived growth factor (PDGF) that become phosphorylated on tyrosine when exposed to PDGF. Exposure of these cells to PDGF also causes an increase in intracellular calcium concentration ([Ca2+]i) and induces a translocation of PKC to the membrane. Preincubation of U-1242 MG cells with several species of gangliosides inhibits the increase in ([Ca2+]i) and PKC translocation in response to PDGF, but GM3 is much less effective than other species tested. This is due to a lack of activation of the receptor tyrosine kinase as monitored by phosphorylation of the receptor on tyrosine residues, but is not due to an inhibition of binding of PDGF to its receptors. The lack of activation of the PDGF receptor tyrosine kinase is due to an inhibition of dimerization of the receptor monomers by gangliosides GM1, GM2, GD1a, GT1b, but not GM3. Therefore, gangliosides may be involved in coordinating the activities of multiple trophic factors simultaneously acting on a cell by regulating the dimerization of their respective receptor monomers.

Mechanisms through which gangliosides inhibit PDGF-stimulated mitogenesis in intact Swiss 3T3 cells: receptor tyrosine phosphorylation, intracellular calcium, and receptor binding.

Several potential mechanisms through which gangliosides could modulate PDGF-stimulated events in Swiss 3T3 cells were studied using intact cells. Of the gangliosides studied, at low micromolar concentrations GM2 was the only one that inhibited PDGF-stimulated DNA synthesis, but GT1b was the most potent between 25 and 100 microM; GM1 was generally the least effective, and GD1a and GM3 had intermediate effects. All gangliosides tested inhibited the PDGF-stimulated increases in free intracellular calcium concentrations ([Ca2+]i) with the rank order of potency being GM1 > or = GT1b > GM2 > GM3. PDGF stimulated phosphorylation on tyrosine of a protein with apparent M(r) = 170 kDa which was immunoprecipitated by an anti-PDGF receptor (beta) antibody, indicating that it is a PDGF receptor. Preincubating the cells with specific gangliosides inhibited tyrosine phosphorylation of this protein in a dose-responsive fashion with the following rank order of potency GD1a = GT1b > GM1 > GM2 > GM3. Autoradiography showed that this was due to a decrease in the proportion of cells synthesizing DNA, and a time study showed that ganglioside did not delay entry of the cells into S phase. These effects were not due to gangliosides interfering with PDGF binding to its receptor because results of competitive binding studies showed that none of the gangliosides studied had an effect on either receptor number or affinity, and did not bind to PDGF in solution. These results show that gangliosides affect several specific components of the complex responses to PDGF in intact cells. The relative effectiveness of individual gangliosides, however, varied among the different cellular and molecular responses. This is interpreted to mean that specific gangliosides modulate to different degrees several molecular mechanisms which converge on the common biological response of mitogenesis, and suggests that gangliosides as a family of molecules may function as coordinators of different molecular events involved in complex cellular processes.