Apoptosis in human glioblastoma cells produced using embryonic stem cell-derived astrocytes expressing tumor necrosis factor-related apoptosis-inducing ligand.
OBJECTIVE
Embryonic stem (ES) cell-derived astrocytes have several theoretical and practical advantages as gene therapy vectors in the treatment of malignant gliomas. The aim of this study was to test the proapoptotic effects of ES cell-derived astrocytes expressing transgenic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in human malignant glioma cells.


METHODS
Mouse ES cells containing a doxycycline-inducible transgene were engineered with human TRAIL (hTRAIL) and then directed to differentiate into astrocytes. The ES cell-derived-TRAIL-expressing astrocytes were cocultured with human malignant glioma cells. Reverse transcriptase polymerase chain reaction, immunocytochemistry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and flow cytometry were used to quantify results. In vitro coculture of ES cell-derived astrocytes expressing hTRAIL with A172 human malignant glioma cells after doxycycline induction caused a significant decrease in cell viability from 85 +/- 2% at baseline to 8 +/- 2% posttreatment (p < 0.001). Labeling with apoptotic markers showed that cell death occurred by means of apoptosis. A significant increase in apoptotic rate (88 +/- 3%) from baseline (4 +/- 2%) was found in A172 cells after doxycycline induction (p < 0.005). This effect was superior to the apoptotic rate seen after treatment with recombinant TRAIL (57 +/- 2%). A decrease in cell viability and an increase in the apoptotic rate were not found in TRAIL-expressing-ES cell-derived astrocytes after induction with doxycycline or in A172 cells exposed to doxycycline alone.


CONCLUSIONS
Engineering of transgenic hTRAIL by using ES cell-derived astrocytes induced apoptosis in human malignant glioma cells while sparing nontumor astrocytes. The apoptotic effects of transgenic hTRAIL are greater than those of recombinant hTRAIL. Analysis of these results suggests that hTRAIL-expressing-ES cell-derived astrocytes should be considered in the development of new in vivo strategies to treat malignant human gliomas.
Germano
Isabelle M
IM
Department of Neurosurgery, Mount Sinai School of Medicine, New York, New York 10029, USA. isabelle.germano@mountsinai.org
Uzzaman
Mahmud
M
Benveniste
Ronald J
RJ
Zaurova
Milana
M
Keller
Gordon
G
D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000235:genetics*; D001253:Astrocytes / Q000502:physiology*; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D004318:Doxycycline; D041722:Genes, Transgenic, Suicide / Q000502:physiology*; D005909:Glioblastoma / Q000473:pathology*; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics*; D051379:Mice; D013234:Stem Cells / Q000166:cytology; D053221:TNF-Related Apoptosis-Inducing Ligand; D023822:Tissue Engineering / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D051017:Apoptosis Regulatory Proteins; D008562:Membrane Glycoproteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; C505654:Tnfsf10 protein, mouse; D014409:Tumor Necrosis Factor-alpha; D004318:Doxycycline