Semi-high throughput method of measuring proteasome inhibition in vitro and in cultured cells.
Summary of "Semi-high throughput method of measuring proteasome inhibition in vitro and in cultured cells."
The ubiquitin proteasome-proteolytic pathway has emerged as one of the most significant pathways in modulating protein homeostasis under both normal and disease states. The use of proteasome inhibitors (PI) has played a pivotal role in understanding protein turn over. The main objective of this work was to develop a comprehensive, fast, and reliable, yet simple in vitro assay that would allow for the identification and characterization of a wide range of PIs. The assays consist of a 96-well plate high throughput (HTP) method to assess proteasome activity in Hs578T breast cancer cell extracts, purified 20S proteasome, using a fluorogenic substrate, Suc-leu-leu-val-tyr-7-AMC, specific to the chymotrypsin-like enzymatic activity of the proteasome. We showed that the chymotrypsin-like activity of the proteasome was inhibited in the two in vitro systems, albeit to different degrees. The assay system also includes two cell-based assays consisting of a vector expressing a fusion protein of green fluorescent protein (gfp) and Mouse Ornithine Decarboxylase (MODC) in Zs578T (parental Hs578T carrying the vector that expresses the fusion protein). In the cell-based assay analyses (qualitatively by microscopy and quantitatively by flow cytometry), treatment of Zs578T with PIs prevented the degradation of MODC, accumulated gfp, indicative of increased proteasome inhibition. Because no single assay represents a definitive proof of proteasome inhibitory activity, combined, these assays should serve as a comprehensive benchmark for the identification and partial characterization of novel inhibitors. In summary, the four-step assay protocol can easily be adapted into a high throughput format to rapidly screen unknown inhibitors.
Affiliation
Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, P. O. Box 66, 1515 Holcombe Blvd., Houston, TX, 77030, USA, kkeyomar@mdanderson.org.
Journal Details
This article was published in the following journal.
Name: Cell biology and toxicology
ISSN: 1573-6822
Pages:
Links
- PubMed Source: http://www.ncbi.nlm.nih.gov/pubmed/20853140
- DOI: http://dx.doi.org/10.1007/s10565-010-9175-1
Medical and Biotech [MESH] Definitions
High-throughput Screening Assays
Rapid methods of measuring the effects of an agent in a biological or chemical assay. The assay usually involves some form of automation or a way to conduct multiple assays at the same time using sample arrays.
Biological Assay
A method of measuring the effects of a biologically active substance using an intermediate in vivo or in vitro tissue or cell model under controlled conditions. It includes virulence studies in animal fetuses in utero, mouse convulsion bioassay of insulin, quantitation of tumor-initiator systems in mouse skin, calculation of potentiating effects of a hormonal factor in an isolated strip of contracting stomach muscle, etc.
Cell Migration Inhibition
Phenomenon of cell-mediated immunity measured by in vitro inhibition of the migration or phagocytosis of antigen-stimulated LEUKOCYTES or MACROPHAGES. Specific CELL MIGRATION ASSAYS have been developed to estimate levels of migration inhibitory factors, immune reactivity against tumor-associated antigens, and immunosuppressive effects of infectious microorganisms.
Benchmarking
Method of measuring performance against established standards of best practice.
Visual Field Tests
Method of measuring and mapping the scope of vision, from central to peripheral of each eye.
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