Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P04637 (p53)
 77,613 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This chapter describes the implementation of the p53-hDM2 protein-protein interaction (PPI) biosensor (PPIB) HCS assay to identify disruptors of p53-hDM2 PPIs. Recombinant adenovirus expression constructs were generated bearing the individual p53-GFP and hDM2-RFP PPI partners. The N-terminal p53 transactivating domain that contains the binding site for hDM2 is expressed as a GFP fusion protein that is targeted and anchored in the nucleolus of infected cells by a nuclear localization (NLS) sequence. The p53-GFP biosensor is localized to the nucleolus to enhance and facilitate the image acquisition and analysis of the PPIs. The N-terminus of hDM2 encodes the domain for binding to the transactivating domain of p53, and is expressed as a RFP fusion protein that includes both an NLS and a nuclear export sequence (NES). In U-2 OS cells co-infected with both adenovirus constructs, the binding interactions between hDM2 and p53 result in both biosensors becoming co-localized within the nucleolus. Upon disruption of the p53-hDM2 PPIs, the p53-GFP biosensor remains in the nucleolus while the shuttling hDM2-RFP biosensor redistributes into the cytoplasm. p53-hDM2 PPIs are measured by acquiring fluorescent images of cells co-infected with both adenovirus biosensors on an automated HCS imaging platform and using an image analysis algorithm to quantify the relative distribution of the hDM2-RFP shuttling component of the biosensor between the cytoplasm and nuclear regions of compound treated cells.
...
PMID:High content screening biosensor assay to identify disruptors of p53-hDM2 protein-protein interactions. 2585 76

High-risk human papillomavirus (HPV) is a common cause of cervical cancer. HPV E6 oncoprotein promotes the degradation of host tumor suppressor gene p53, leading to the development of tumors. Therapeutic strategies that specifically target E6, which is constitutively expressed in tumors and is not present in normal tissues, may be highly effective and safe. CRISPR-CRISPR associated protein 9 (Cas9) is one of the genome editing technologies that has recently garnered attention, and is used to knockout target gene expression. By combining cervical cancer cell lines engineered to constitutively express Cas9 and an adeno-associated virus (AAV) vector carrying a single guide (sg) RNA targeting E6 (AAV-sgE6), the present study sought to investigate the effects of this novel therapeutic approach on cervical cancer. The Cas9 gene was transfected into three high-risk HPV-positive cervical cancer cell lines (HeLa, HCS-2, and SKG-I) to establish cell lines that constitutively expressed Cas9. Using these cell lines, genetic mutations and their frequencies, as well as the levels of protein expression, apoptosis and cell proliferation were examined in vitro. In addition, the effects of AAV-sgE6 were examined in a mouse model of cervical cancer in vivo by a single administration of AAV-sgE6 directly into subcutaneous tumors. The results demonstrated that multiple mutations occurred frequently in the targeted E6 genomic sequence in cervical cancer cells transduced with AAV-sgE6. In addition, these AAV-sgE6-transduced cells had reduced expression of E6, increased expression of p53, increased apoptosis and their growth was suppressed in a concentration-dependent manner. Furthermore, subcutaneous tumor growth was significantly suppressed in vivo following intratumoral administration of AAV-sgE6, and adverse events due to AAV-sgE6 administration were not observed. Collectively, the present results indicated that targeting E6 expression in high-risk HPV by CRISPR-Cas9 is a highly specific and effective strategy that may be effective in treating patients with cervical cancer.
 ...
PMID:CRISPR/Cas9-mediated cervical cancer treatment targeting human papillomavirus E6. 3067 84