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

Introduction

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. Using flow cytometry, we selected glioma cells (U1242MGGD3 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.

Objectives

To investigate the biological effects of the expression of endogenous GD3; to investigate the molecular mechanisms of apoptosis in the U-1242MG human glioma cell line; and to investigate the molecular mechanisms through which GD3 induces apoptosis;

Methods

Cell culture: Parental U-1242 MG cells were grown in minimal essential medium with 10% calf serum supplemented with penicillin/streptomycin (80 U/mL and 80 lg/mL) and Fungizone (0.22 lg/mL). Cells transfected with Tet-system vectors were grown in minimal essential medium supplemented with the same antibiotics in 10% Tet-system fetal bovine serum. Plasmid construction, TetOn tri-cistronic construct, GD3 synthase construct and Generation of stably transfected cell lines: We transfected U-1242MG with these two constructs. We selected U42-GRD2 cells on the basis of high GD3 expression under the effect of doxycycline. Detection of GD3 expression using: Flow cytometry; reverse transcription–polymerase chain reaction; immunocytochemistry and chemical analysis. Effects of GD3 expression on cell growth: Apoptosis assays; caspase-8 inhibition; Western immunoblotting; and determination of proliferative index.

Results

Modification of the TetOn system; detection of doxycycline-induced GD3 expression using the TetOn system:

Thin layer chromatographic separation of gangliosides and neutral glycolipids: (a) U1242MG-GD3 and U1242MG-EV cells were treated with 10 lg/mL doxycycline (a, lanes 1 and 3; b, lanes 1 and 3) and without Dox (a, lanes 2 and 4; b, lanes 2 and 4) for 6 days, after which total glycolipids were isolated and analyzed.

Endogenous GD3 induces apoptosis in U1242MG-GD3. U1242MG-GD3 and U1242MG-EV cells were exposed to 0 or 10 lg/mL doxycycline (Dox) for 6 days, then either (a) incubated with Annexin V conjugated to Alexa Fluor 647, or (b) a TUNEL assay was performed.

Flow cytometric analysis for enhanced green fluorescent protein (EGFP) can be used as an indicator of apoptosis. The arrow indicates U1242MG-GD3 cell population with low EGFP expression. B) GD3-induced apoptosis is mediated by caspase-8.

Doxycycline treatment of U1242-GD3 cells decreases the proliferative index. U1242-GD3 cells were grown with and without doxycycline for 6 days, at the end of which they were exposed first to MIB-1 antibody and then to Alexa 647- conjugated secondary antibody. Green fluorescent positive cells express the Tet-On gene, and provide an estimate of the total cell number.

Discussion

The present study was designed to determine if endogenous GD3 induces apoptosis in U-1242 MG glioma cells. To investigate this we used a newly modified TetOn system, which enabled us to strictly regulate the expression of GD3. From the cells originally infected with these constructs, we selected a population of cells with the maximum controllable Dox regulated GD3 expression using flow cytometry. Fluorescence intensity from Alexa Fluor 647 in these cells indicates that U1242MG-GD3 cells express high levels of GD3. U1242MGGD3 cells exhibited morphological changes of apoptosis after 4–6 days exposure to Dox. GD3-induced apoptosis was also confirmed using the TUNEL assay. Here we report that GD3-induced apoptosis in U-1242 MG cells via activation of a caspase-8. Collectively, these findings are compelling evidence that increased levels of endogenously synthesized GD3 induce apoptosis in U1242MG-GD3 cells.

We conclude that GD3 synthesis in U-1242 MG human glioma cells can be controlled by our modified TetOn system. Increased expression of GD3 synthase caused U-1242 MG cells to undergo apoptosis determined by cytological appearance, increased annexin V binding, DNA fragmentation, and decreased expression of EGFP. Regulated GD3 expression using this system will be extremely useful in future studies on the mechanism of GD3-induced apoptosis in human glioma cells.