Reactivation of death receptor 4 (DR4) expression sensitizes medulloblastoma cell lines to TRAIL.
OBJECTIVE
Apoptosis, a key cellular response to therapeutic agents is often inactivated in tumor cells. In this study, we evaluated the expression of the tumor necrosis family of death receptors, DR4 and DR5, in medulloblastoma tumor samples and cell lines to determine if epigenetic modulation of gene expression could sensitize tumor cell lines to TRAIL-mediated apoptosis.


METHODS
Human medulloblastoma samples and cell lines were analyzed for DR4 and DR5 expression by quantitative PCR and immunofluorescence assays. Cell lines with downregulated expression of one or both genes were treated with the histone deacetylase inhibitor, MS-275, and the expression of DR4 and DR5 measured by quantitative PCR, Western blotting, flow cytometry and chromatin immunoprecipitation assays. Induction of apoptosis in the presence of MS-275 was evaluated by TUNEL assay and its ability to augment TRAIL-mediated cytotoxicity was determined by MTT assays, Western blotting and flow cytometry.


RESULTS
Compared to normal cerebellum, DR4, but not DR5 expression was consistently downregulated in medulloblastoma tumor samples and in Daoy and D283 cell lines. Interestingly, MS-275 decreased cell growth and induced apoptosis in Daoy and D283 cells. In Daoy cells, this coincided with increased histone H3 and H4 acetylation at the DR4 promoter and enhanced DR4 gene and protein expression as well as elevated Caspase-8 activity. The involvement of DR4 in the cellular response to MS-275 was further confirmed by the observation that knockdown of DR4 and FADD abrogated apoptosis. Further, addition of TRAIL to MS-275 treated cells resulted in an enhancement of apoptosis, suggesting that the upregulated death receptors were functional.


CONCLUSIONS
Our study provides an understanding of the role of DR4 in apoptosis of medulloblastoma cell lines and suggests a potential contribution of aberrant histone deacetylation to the resistance of medulloblastoma cells to therapeutic death.
Aguilera
Dolly G
DG
Department of Pediatrics, University of Texas, M.D. Anderson Cancer Center, Houston, 77030, USA.
Das
Chandra M
CM
Sinnappah-Kang
Neeta D
ND
Joyce
Celine
C
Taylor
Pete H
PH
Wen
Sijin
S
Hasselblatt
Martin
M
Paulus
Werner
W
Fuller
Greg
G
Wolff
Johannes E
JE
Gopalakrishnan
Vidya
V
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001549:Benzamides / Q000494:pharmacology; D015153:Blotting, Western; D053181:Caspase 8 / Q000378:metabolism; D045744:Cell Line, Tumor; D002528:Cerebellar Neoplasms / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053200:Fas-Associated Death Domain Protein / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D047468:Immunoprecipitation; D020287:In Situ Nick-End Labeling; D008527:Medulloblastoma / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
D001549:Benzamides; D004791:Enzyme Inhibitors; D053200:Fas-Associated Death Domain Protein; D056572:Histone Deacetylase Inhibitors; D011725:Pyridines; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; C118739:entinostat; D053181:Caspase 8