Differential activation of the Fas/CD95 pathway by Ad-p53 in human gliomas.
Adenoviral p53 gene transfer (Ad-p53) induces apoptosis in glioma cells expressing mutant p53, but fails in cells with wild-type p53. Endogenously, gliomas express varied levels of Fas/CD95, yet constitutively high levels of Fas/CD95 ligand. Because the mechanism behind the differential apoptotic response to Ad-p53 infection remains elusive, we examined how the Fas/CD95 pathway is involved in U87MG (wt-p53), D54 (wt-p53), U251MG (mutant-p53), and U373MG (mutant-p53) glioma cell lines. Ad-p53 infection did not alter the levels of Fas/CD95 ligand in either wild-type or mutant p53-expressing cell lines. In contrast, Ad-p53 infection led to an approximately 3-fold increase in Fas/CD95 mRNA expression in mutant p53-bearing cell lines but not in their wild-type (wt) counterparts, as assessed in an RNase protection assay. Fas/CD95 mRNA induction appeared to be regulated at the transcriptional level because Ad-p53 infection resulted in up to a 4-fold increase in Fas/CD95 promoter reporter activity. Subsequently, flow cytometric analysis revealed a 2- to 4-fold increase in surface Fas/CD95 expression following Ad-p53 infection in mutant-p53-containing cell lines. Use of the protein transport inhibitor Brefeldin A significantly inhibited Ad-p53-induced surface Fas/CD95 expression, but only partially inhibited apoptosis in mutant-p53 cell lines. These results suggest that p53 regulates Fas/CD95 expression at the transcriptional level and through protein trafficking in mutant-p53 cell lines. Fluorogenic activity assays demonstrated that induction of caspase-8 activity following Ad-p53 infection correlated with increases in Fas/CD95 expression. Incubating cells with a caspase-8-specific inhibitor Ac-IETD-CHO prior to Ad-p53 infection inhibited caspase-8 activity and apoptosis. Together, our results suggest that regulation of the Fas/CD95 pathway is partly responsible for Ad-p53-induced apoptosis in glioma cells, which depends on the p53 status of the involved cells. Additionally, the inability of Ad-p53 to activate the Fas/CD95 pathway in wt-p53 glioma cells coincides with their apoptotic-resistant phenotype. Further elucidation of the nature of this resistance could ultimately augment the efficacy of Ad-p53 gene therapy.
Cerrato
Julie A
JA
The Brain Tumor Center, Department of Neuro-Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
Khan
Tahira
T
Koul
Dimpy
D
Lang
Frederick F
FF
Conrad
Charles A
CA
Yung
W K Alfred
WK
Liu
Ta-Jen
TJ
D000256:Adenoviridae / Q000235:genetics*; D017209:Apoptosis; D001932:Brain Neoplasms / Q000378:metabolism*; D020126:Brefeldin A / Q000494:pharmacology; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival; D053222:Fas Ligand Protein; D005434:Flow Cytometry; D017930:Genes, Reporter; D016158:Genes, p53; D005910:Glioma / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism; D009154:Mutation; D010641:Phenotype; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D014162:Transfection; D019014:fas Receptor / Q000096:biosynthesis*
C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D019014:fas Receptor; D020126:Brefeldin A; D012260:Ribonucleases; C505530:CASP8 protein, human; D053181:Caspase 8; D020169:Caspases