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.
Saqr
H E
HE
Department of Pathology, The Ohio State University, Columbus, Ohio 43210, USA.
Omran
O
O
Dasgupta
S
S
Yu
R K
RK
Oblinger
J L
JL
Yates
A J
AJ
D000469:Alkaline Phosphatase / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology*; D001426:Bacterial Proteins / Q000502:physiology; D002352:Carrier Proteins / Q000502:physiology; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002855:Chromatography, Thin Layer / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D005732:Gangliosides / Q000378:metabolism / Q000502:physiology*; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000502:physiology*; D005910:Glioma / Q000378:metabolism / Q000473:pathology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D020287:In Situ Nick-End Labeling / Q000379:methods; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013997:Time Factors; D014162:Transfection / Q000379:methods
D001426:Bacterial Proteins; D002352:Carrier Proteins; D005732:Gangliosides; D012333:RNA, Messenger; C083139:Tet O resistance protein, Bacteria; D049452:Green Fluorescent Proteins; C026226:ganglioside, GD3; D000469:Alkaline Phosphatase; C505530:CASP8 protein, human; D053181:Caspase 8; D020169:Caspases; D004318:Doxycycline