Doxorubicin induces apoptosis and CD95 gene expression in human primary endothelial cells through a p53-dependent mechanism.
Regulation of the homeostasis of vascular endothelium is critical for the processes of vascular remodeling and angiogenesis under physiological and pathological conditions. Here we show that doxorubicin (Dox), a drug used in antitumor therapy, triggered a marked accumulation of p53 and induced CD95 gene expression and apoptosis in proliferating human umbilical vein endothelial cells (HUVECs). Transfection and site-directed mutagenesis experiments using the CD95 promoter fused to an intronic enhancer indicated the requirement for a p53 site for Dox-induced promoter activation. Furthermore, the p53 inhibitor pifithrin-alpha (PFT-alpha) blocked both promoter inducibility and protein up-regulation of CD95 in response to Dox. Up-regulated CD95 in Dox-treated cells was functional in eliciting apoptosis upon incubation of the cells with an agonistic CD95 antibody. However, Dox-mediated apoptosis was independent of CD95/CD95L interaction. The analysis of apoptosis in the presence of PFT-alpha and benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone revealed that both p53 and caspase activation are required for Dox-mediated apoptosis of HUVECs. Finally, Dox triggered Bcl-2 down-regulation, cytochrome c release from mitochondria, and the activation of caspases 9 and 3, suggesting the involvement of a mitochondrially operated pathway of apoptosis. These results highlight the role of p53 in the response of primary endothelial cells to genotoxic drugs and may reveal a novel mechanism underlying the antitumoral properties of Dox, related to its ability to induce apoptosis in proliferating endothelial cells.
Lorenzo
Elisa
E
Centro de Biologia Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas, Universidad Autónoma de Madrid, Facultad de Ciencias, Madrid, Spain.
Ruiz-Ruiz
Carmen
C
Quesada
Antonio Jesús
AJ
Hernández
Gabriela
G
Rodríguez
Antonio
A
López-Rivas
Abelardo
A
Redondo
Juan Miguel
JM
D017209:Apoptosis / Q000187:drug effects*; D001483:Base Sequence; D052160:Benzothiazoles; D002455:Cell Division; D002478:Cells, Cultured; D017931:DNA Primers; D004317:Doxorubicin / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D011401:Promoter Regions, Genetic; D013844:Thiazoles / Q000378:metabolism; D014050:Toluene / Q000031:analogs & derivatives / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*; D019014:fas Receptor / Q000235:genetics*
D052160:Benzothiazoles; D017931:DNA Primers; D013844:Thiazoles; D016159:Tumor Suppressor Protein p53; D019014:fas Receptor; D014050:Toluene; D004317:Doxorubicin; C121565:pifithrin