Live-cell imaging of caspase activation for high-content screening Journal Article
| Authors: | Antczak, C.; Takagi, T.; Ramirez, C. N.; Radu, C.; Djaballah, H. |
| Article Title: | Live-cell imaging of caspase activation for high-content screening |
| Abstract: | Caspases are central to the execution of programmed cell death, and their activation constitutes the biochemical hallmark of apoptosis. In this article, the authors report the successful adaptation of a high-content assay method using the DEVDNucView488ĝ.,¢ fluorogenic substrate, and for the first time, they show caspase activation in live cells induced by either drugs or siRNA. The fluorogenic substrate was found to be nontoxic over an exposure period of several days, during which the authors demonstrate automated imaging and quantification of caspase activation of the same cell population as a function of time. Overexpression of the antiapoptotic protein Bcl-XL, alone or in combination with the inhibitor Z-VAD-FMK, attenuated caspase activation in HeLa cells exposed to doxorubicin, etoposide, or cell death siRNA. This method was further validated against 2 well-characterized NSCLC cell lines reported to be sensitive (H3255) or refractory (H2030) to erlotinib, where the authors show a differential time-dependent activation was observed for H3255 and no significant changes in H2030, consistent with their respective chemosensitivity profile. In summary, the results demonstrate the feasibility of using this newly adapted and validated high-content assay to screen chemical or RNAi libraries for the identification of previously uncovered enhancers and suppressors of the apoptotic machinery in live cells. © 2009 Society for Biomolecular Sciences. |
| Keywords: | controlled study; unclassified drug; human cell; doxorubicin; erlotinib; antineoplastic agents; cell proliferation; cell death; cell survival; gene overexpression; apoptosis; models, biological; etoposide; lung non small cell cancer; small interfering rna; cancer cell culture; enzyme activation; high throughput screening; drug screening assays, antitumor; caspase; chemical ht screening; high-content assay; live cells; rnai ht screening; aspartylglutamylvalyl aspartic acid; benzyloxycarbonylvalylalanylaspartyl fluoromethyl ketone; caspase inhibitor; peptide; protein bcl xl; cell population; chemosensitivity; enzyme substrate; gene library; hela cell; molecular imaging; amino acid chloromethyl ketones; bcl-x protein; caspases; cell line, tumor; cysteine proteinase inhibitors; diagnostic imaging; hela cells; high-throughput screening assays; transfection |
| Journal Title: | Journal of Biomolecular Screening |
| Volume: | 14 |
| Issue: | 8 |
| ISSN: | 1087-0571 |
| Publisher: | Sage Publications |
| Date Published: | 2009-09-01 |
| Start Page: | 956 |
| End Page: | 969 |
| Language: | English |
| DOI: | 10.1177/1087057109343207 |
| PUBMED: | 19726787 |
| PROVIDER: | scopus |
| PMCID: | PMC3613133 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 9" - "Export Date: 30 November 2010" - "CODEN: JBISF" - "Source: Scopus" |
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