ABSTRACT
The p53 tumor suppressor belongs to a gene family that includes two other structurally and functionally related members: p73 and p63. The regulation of p53 activity differs significantly from that of p73 and p63. To enhance the tumor suppressive activity of p53, we constructed six recombinant adenoviruses that encode hybrid proteins with three functional domains derived from either p53 or TAp63γ. The potency of these hybrid molecules in suppressing tumorigenesis was evaluated using in vitro and in vivo models. Of the hybrid molecules tested, one hybrid named p63-53O was the most potent activator of apoptosis in human cancer cells. The p63-53O hybrid is composed of the transcriptional activation domain and DNA-binding domain of TAp63γ and the oligomerization domain of p53. The p63-53O hybrid efficiently transactivated p53AIP1. Moreover, silencing of p53AIP1 partially abolished the apoptotic response to p63-53O in human cancer cells. The p53-p63 hybrid molecule is a novel potent anti-proliferative agent for the treatment of cancer.
Subject(s)
Gene Expression Regulation, Neoplastic , Genes, p53 , Neoplasms/therapy , Recombinant Fusion Proteins/therapeutic use , Adenoviridae/genetics , Adenoviridae/metabolism , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacology , Apoptosis , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Cell Line, Tumor , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Female , Genetic Vectors/genetics , Genetic Vectors/metabolism , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasms/genetics , Neoplasms/metabolism , Protein Structure, Tertiary , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Activation , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Xenograft Model Antitumor AssaysABSTRACT
The mitotic checkpoint gene CHFR (checkpoint with forkhead and ring finger domains) is silenced in various human cancers by promoter hypermethylation, suggesting that CHFR is a tumor suppressor. Here, we show that CHFR functions as a negative regulator of the nuclear factor-kappaB (NF-kappaB) pathway. Expression of CHFR inhibited NF-kappaB reporter activity, whereas knockdown of CHFR activated reporter activity. These activities are independent of its RING finger domain. Furthermore, we found that CHFR physically interacts with p65 in cells. Electrophoretic mobility shift assays (EMSAs) and ELISA-based NF-kappaB-binding assays showed that CHFR negatively regulated transcriptional activity of p65. In addition, our data show that interleukin (IL)-8 is significantly downregulated by CHFR, and that the migration of human endothelial cells is suppressed in culture medium conditioned from CHFR-expressing cancer cells. Using a xenograft model, we show that neovascularization is suppressed by adenovirus-mediated transfer of CHFR. These results indicate that expression of CHFR markedly reduces the expression of IL-8 through the inhibition of NF-kappaB. As the NF-kappaB signaling pathway plays a critical role in the development and progression of cancer, our findings show the functional relationship between epigenetic alteration and inflammation/angiogenesis in human cancer cells, thereby showing several potential targets for therapeutic intervention.
